Team:Leicester/September2012

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(11:00 am) Some of the team calculated the concentration of 16s extracted DNA to be run on an agarose gel and later for sequencing. The calculations allow the most efficient use of the DNA stock.

(14:00 pm) 2μl of DNA were added for samples 3, 4, 5 and 6 whereas 4μl of sample 2 DNA were added due to the stock concentration being lower. The amount of marker added was varied, which allowed the concentration of samples to be determined and the accuracy of the nano-drop checked.

(15:45 pm) The gel was loaded as:

X

X

Marker (4μl)

2 (4μl)

3 (2μl)

4 (2μl)

Marker (2μl)

5 (2μl)

6 (2μl)

Marker (1μl)

X

X

X

X

The gel was then run for 1 and a half hours.

(16:30 pm) A group meeting was held to inform the supervisors of how the project has progressed and discuss a detailed plan for the 4th September. Plans for the future of the project were also discussed.

(17:30 pm) The gel was then transilluminated (pictured below) and the concentration of the DNA was determined for use in sequencing tomorrow (4th September 2012).

(09:00 am) Although some colonies from the growth curve experiment could be counted, but in general there was too much growth.

(09:15 am) The group worked on the wiki for the rest of the day writing the past weeks work in, making sure all the details were correct and in the right order, and then attributing the members as required.

(09:00 am) In preparation for a meeting later in the day, members of the lab discussed pathways involved in polystyrene degradation.

(11:00 am) A meeting was held with supervisors discussing the next steps of the project, with hopes to try and engineer a BioBrick. It is suspected that genes involved in toluene degradation such as TodX, TodC1&2, TobA&B and others present in the operon may also be involved in the pathway for polystyrene degradation.

(12:00 pm) Databases containing genomic sequencing and BLAST were searched to find bacteria that used proteins similar to those discussed above for PCR. Although Pseudomonas aeruginosa didn’t have any of the genes, Pseudomonas putida F1 had them all.

(15:00 pm) With the help of a supervisor, one member of the team set up a PCR reaction for 16S DNA to increase the amount of DNA in the forward and reverse directions (in separate tubes) ready for sequencing tomorrow.

Yellow and Orange colonies were plated out and 01#502 was streaked again. This allows fresh colonies to be maintained, ready for storage at the end of the project.

(16:00 pm) The wiki was updated and searching BLAST for proteins continued.

(09:00 am) An early start with no set experiment today. Two of the colonies were plated out to see if they are two forms of the same bacteria or simply two separate species. This required plates being made, then individual colonies picked out to streak them. Once finished it is a day working on modelling, writing protocol and other computer work before checking the results of the plates in the late afternoon.

(16:00 pm) Some spec readings were taken of the MMP broth, with the blank just being MM broth. At 600nm wavelength the results came back for the mixed culture as 0.334, and the orange culture as 0.6.

(14:00 pm) A meeting was held to share the achievements of each individual section of the project (modelling, lab work and chemistry). Job roles were assigned to each person to complete in labs and on the computer modelling.

(16:30 pm) More polystyrene minimal media plates were prepared, ready for plating out some colonies from CSE kits, to see whether more positive results can be obtained from other samples.

(17:00 pm) Minimal media was made from minimal broth.

(17:05 pm) The Boilate was set up with a sample of our unknown bacteria to extract the DNA, ready to set an overnight PCR reaction so that we can sequence the 16S ribosome later on this week.

(17:20 pm) The master mix was prepared for 14 PCR reactions. 270μl of the master mix was made up, enough for aliquoting 18μl into 15 reactions.

The reaction mixture contained:

171μl PCR H₂0
60μl HF buffer
6μl dNTP's
15μl Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15μl Primer B 519R GWATTACCGCGGCKGCTG
Template DNA to be added later as it is not PCR clean
3μl DNA Pol

DNApol is stored in glycerol and required mixing and spinning down for a second. The remainder of the dNTPs were disposed of as it cannot be freeze thawed. In the PCR hood, the master mix was aliquoted into 9 separate PCR eppendorfs with 18μl in each and the negative control prepared using PCR clean H20. The tubes were then taken to the bench where the DNA and diluted bacteria were added. 2μl of all samples were added to make a final volume of 20μl, including the positive and negative controls (see gel lane organization).

PCR Cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)

Also the teams organic chemists have been designing a number of mechanisms. The mechanism below shows the final synthesis after much improvements. It shows the conversion of the monomer styrene being converted to lactic acid. A company called styrofoam specialise in polymerising lactic acid for easy environental breakdown. We think this route may be a viable synthetic route however due to the nature of the reagents used and conditions, there are other routes to look in to.

(09:00 am) A 2% agarose gel was prepared to run the PCR products on. One member of the team updated the project Wiki page. Attempts were made to track down the P. putida F1 strain which has possibly been located.

(09:40 am) The gel was poured and left to set. 5μl of loading dye was added to the PCR products ready to run on the gel.

(11:15 am) The gel was loaded and run at 120 volts. Lane organisation:
1) Positive control 10ng P. aeruginosa DNA from the Maxwell prep
2) Positive control 10ng P. aeruginosa DNA from the Maxwell prep
3) Neat orange culture DNA
4) x10^-1 dilution Orange culture DNA
5) x10^-2 dilution Orange culture DNA
6) x10^-3 dilution Orange culture DNA
7) x10^-4 dilution Orange culture DNA
8) Neat Yellow culture DNA
9) x10^-1 dilution Yellow culture DNA
10) x10^-2 dilution Yellow culture DNA
11) x10^-3 dilution Yellow culture DNA
12) 5μl Marker 100bp thermo scientific
13) Negative Bench H₂0
14) Negative Hood PCR H₂0

The Sau3AI partial digest was prepared. The experiment was run for 30 minutes as from the last test the only lanes which were digested enough were the times 5-15 minutes. A "no enzyme" control was also run.

(12:30 pm) Following the results of sequencing the 16S ribosomal DNA, a BLAST Search was conducted to find the genus of the bacteria cultured from the 01#502 CSE kit, which turned out to be a Pseudomonas of unknown species.

(13:20 pm) The agarose gel was stopped and a photo obtained from the transilluminator:

The PCR was repeated overnight with the x10-1 orange dilution and the 0 yellow dilution with 5μl of each as the amount recovered from this gel may not be enough for the sequencing. There were two different coloured bacteria growing and this was conducted to distinguish if these are two different species of bacteria or the same one with a different morphology of colony.

To see how much DNA can be recovered, a QIAGEN gel extraction was performed and more DNA amplified overnight.

A gel was prepared for the Sau3AI digest which has completed.

(15:10 pm) The gel was cut and the samples of DNA removed and weighed. The QG buffer was added at x6 the amount in μl of the amount of agarose there was. The samples were placed in the 50 degree incubator and vortexed every 2 minutes to dissolve the agarose.

(15:30 pm) Unfortunately, the Sau3AI digest was carried out at 37 degrees rather than the intended room temperature which might have produced better bands. Therefore, the experiment was repeated. However, the gel was still run to obtain results and confirm the need to reduce the activity of the enzyme. Spectrophotometric analysis was conducted for the experiment set up on the 31st, after 2 day's extra growth; mmb with 5% poly resulted in OD600 mixed = 0.053 at a 10x dilution, 0.53 at 1x dilution. Orange was 0.072 at a x10 dilution, 0.72 with no dilution. This was higher than previous demonstrating that the bacteria was growing in the broth with no other carbon other than sugar beads at 5% concentration. To confirm this the experiment, it will be repeated with more controls totalling at 8 tubes.

(16:00 pm) A gel was loaded using the Sau3AI digest from earlier, the DNA was extracted from the earlier electrophoresis using a QIAGEN QIAquick Gel Extraction Kit and the wiki protocol was updated.

(16:30 pm) The Boilate was prepared for PCR overnight using the 16S yellow and orange colonies. Only the 5 neat yellow cultures and 5 x10-1 orange cultures will undergo PCR as these seemed to be the best. Unfortunately, it seems that there will not be enough DNA to sequence.

(17:15 pm) The Boilate was finished: centrifuging and then removing the supernatant. Dilutions were prepared while the master mix for PCR was prepared in the PCR hood.

(17:30 pm) The Sau3A1 digest was then transilluminated:

(17:45 pm) The DNA was added to the samples ready for PCR. The program used was the iGEM16S which progresses as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever (this is for the end of the reaction).

(09:00 am) A new 2% gel was prepared for the PCR samples which were being prepared. Prep for the Sau3A1 digest at room temperature began.

(10:30 am) One of the gels was remade, as well as another 2% gel ready for the "Gelception" gel, where the team is testing the extraction method.

(12:10 pm) The PCR products were then loaded onto a gel using as much of the sample as possible (22μl). The gel was run for 2.5 hours at 120 Volts, ensuring the bands are well separated for gel extraction. The Sau3A1 prep was finished and samples placed into the freezer.

(13:00 pm) Some lab members looked at the primer designs for BioBrick.

(15:35 pm) The primers were designed, ready for ordering Monday morning. Once the gel finished, it was taken down for transilluminating. Some plates were also taken as some Pseudomonas species' fluoresce under UV light, as can benzene rings. Below is the photo produced from the gel.

(16:00 pm) None of the plates fluoresce under UV which may have been due to the excitation wavelength being different from those attempted. The PCR gel was then wrapped up and stored for gel extraction next week. As the Nanodrop results were low for yesterday's gel extraction from the PCR (at 4.8 for Orange, 5.1 for mixed and 3.2 for yellow when the two extracts were combined), PCR will be conducted using these samples to increase the amount of DNA. Time constraints meant that the "Gelception" gel is to be run on Monday.

(16:30 pm)The finalised primer sequences were sent to a supervisor to be checked and ordered. The master mix for PCR was set up in the PCR hood.There will be 13 PCR reactions, requiring 270µl of the master mix, enough for aliquots of 18µl in 15 reactions. Reaction mixture list:
171µl PCR H20
60µl HF buffer
6µl dNTPs
15µl Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15µl Primer B 519R GWATTACCGCGGCKGCTG
2µl Template DNA to be added later to each PCR tube as it is not PCR clean
3µl DNA Polymerase

DNApol is stored in glycerol and required mixing and spinning down for a second. The remainder of the dNTPs were disposed of as it cannot be freeze thawed. In the PCR hood, the master mix was aliquoted into 9 separate PCR eppendorfs with 18μl in each and the negative control prepared using PCR clean H20. The tubes were then taken to the bench where the DNA and the positive P.aeruginosa DNA and negative bench H2O controls. All samples were added at 2µl to make a final volume of 20µl.

PCR cycle program used was iGEM16S (same as before):

This time instead of loading bacteria from a boilate reaction, a 1/100 dilution was added in of the three different gel extractions at 2µl to each tube to amplify the amount of DNA in these samples. The 1/100 samples were prepared by taking 1µl of the DNA and adding in 99µl of TE buffer. Reaction organisation:
1 = Positive P. aeruginosa control
2 = Orange 100x dilution
3 = Orange 100x dilution
4 = Orange 100x dilution
5 = Yellow 100x dilution
6 = Yellow 100x dilution
7 = Yellow 100x dilution
8 = Yellow 100x dilution
9 = Mixed 100x dilution
10 = Mixed 100x dilution
11 = Mixed 100x dilution
12 = Negative control bench
13 = Negative control hood

Yellow had the lowest stock concentration so four reactions were prepared. Due to the difficulties with the dilutions in the boilate from Wednesday, multiple reactions were run for each of the extractions in case one doesn't work.

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(09:00 am) A 0.7% gel was prepared and DNA markers found - ”HyperLadder 1".

(10:30 am) A 2% agarose gel was loaded with DNA recovered from a previous gel from last week. The lane order was as follows:
5µl DNA HyperLadder 1
x
4µl 100bp DNA ladder
x
20µl of DNA recovered from PCRed mixed colonies
x
2µl DNA HyperLadder 1
x
20µl of DNA recovered from PCRed Orange colonies
x
20µl of DNA recovered from PCRed Yellow colonies
x
1µl DNA HyperLadder 1
x

This gel was run at 120 volts.

(11:00 am) The 0.7% gel was poured ready for running the Sau3A1 digests.

(11:50 am) The gel containing recovered DNA from a previous gel was transilluminated. It showed little band movement and the gel was run for an additional hour.

(12:15 pm) The Sau3A1 digests were loaded into wells on the set 0.7% gel. The lane organisation was as follows:
x
DNA HyperLadder 1
x
DNA digest without enzyme
DNA digest after 0 minutes
DNA digest after 5 minutes
DNA digest after 10 minutes
DNA digest after 15 minutes
DNA digest after 20 minutes
DNA digest after 25 minutes
DNA digest after 30 minutes
x
DNA HyperLadder 1
x

This will be run at 100 volts

(13:00 pm) The recovered DNA gel was retransilluminated, and the bands are a lot more spread:

(13:20 pm) The Sau3A1 digest was checked and it was decided to leave it in for longer, though the loading dye appeared to be running slightly quicker at one end of the gel.

(13:30 pm) The primers that were designed on Friday were looked at by one of the supervisors. The adjustments add degeneracy to make them more likely to PCR out the genes that the team want to extract. BLAST searches of the FASTA data, showed that the sequence in P. putida, regardless of strain, is remarkably conserved, with only one or two bases needing any degeneracy at primer binding sites. These modified primers were sent off to a supervisor in advance of the deadline and will hopefully arrive at the end of the week, allowing PCR to begin on the genes that will be made into Biobricks.

(14:45 pm) Once the Sau3A1 digest gel showed the loading dye was a couple of inches off the end, it was taken to the transilluminator. The results are depicted below.

(09:00 am) P. putida was streaked out onto LA to isolate single colonies. The P. putida strains were also put into LB for Maxwell prep tomorrow to extract the DNA. The 1/100 dilution PCR was prepared for running on a gel that had been stored in the fridge. The wiki was updated. A 2% gel was prepared for running the PCR reaction products.

(11:00 am) Polystyrene 'sugar' was prepared for a polystyrene-propane experiment to see whether propane, which is an expanding agent in the expansion of polystyrene, could have caused the mystery Pseudomonas species to grow rather than the polystyrene. Propane is already in the sugar before expansion and requires ventilating it for several days to remove the majority of the propane. However, if the lid is left on for a time, propane will still build up. The 'sugar' was soaked in varying concentrations of propane, mixed with minimal media broth up to 0.4 ml, completely covering the 0.5g 'sugar' that will be put in each agar dish tomorrow. 0.4, 0.2, 0.1, 0.05 and 0.00ml of propane were used to soak 0.5g of polystyrene 'sugar' overnight on a shaker at 275RPM.

The Rockethub site for the project was edited in order to make it easier to read.

(14:30 pm) The gel was loaded with 1/100 dilution the PCR reaction. Unfortunately, with one of the gels, the comb was too long and the sample went straight through, wasting the majority of the first two samples.

Lane organisation:
1 = P. aeruginosa positive control
2 = Orange 100x dilution
3 = Orange 100x dilution
4 = Orange 100x dilution
5 = Yellow 100x dilution
6 = Yellow 100x dilution
7 = Yellow 100x dilution
8 = Yellow 100x dilution
9 = Mixed 100x dilution
10 = Mixed 100x dilution
11 = Mixed 100x dilution
DNA Hyperladder 5ul
13 = Negative control bench
14 = Negative control hood

This gel was run 2.5 hours at 120V . Each sample was 20μL, except samples 1 and 2 of which only 2μL and 5μL was left respectively.

(15:00 pm) Some Corning broth solutions were made using the pentane, 'sugar' and bacteria. They were made with varying amounts of pentane: 0.00, 0.05, 0.10, 0.20 or 0.40ml. There were 4 reaction conditions prepared: with the yellow Pseudomonas culture, 0.5g of polystyrene 'sugar', with the yellow bacteria only, with 0.5g polystyrene 'sugar' only, and with neither polystyrene or bacteria. The final volume for all solutions was made up to 10ml with the addition of minimal salts medium broth. Along with the 'sugar' soaking in pentane, this was put in the shaker at 240rpm overnight at room temperature to allow time to grow. It is planned to spec analyse the broth tomorrow and if the set with polystyrene and bacteria have the same growth as those without polystyrene, but with bacteria, then pentane has little if any effect on the growth of the yellow colony.

(15:40 pm) The bacteria are loaded into the Pentane Poly experiment, while the gel extraction of the PCR reaction of the orange and yellow single 16S was done. The experiment with the Yellow, Mixed and Orange colonies were ran through a spectrophotometer, however this experiment is going to be done again in triplicate to make sure that the results are not random, as well as the pentane with poly experiment to rule out this.

(16:00 pm) The yellow bacteria was inoculated in 10 corning tubes for the pentane test.

(17:00 pm) The gel extraction is completed at the same time a gel is being made ready for tomorrow. There were a total of 22 extractions going on:

1-5 were the orange bacteria.

6-10 were the yellow bacteria which have now been combined so there is only 3 tubes to Nanodrop tomorrow, which is equal to three lanes on the gel.

11-13 were orange PCR product gel extraction x100 dilution PCR product's.

14-17 were yellow PCR product gel extraction 100x dilution PCR product’s.

18-20 were the mixed PCR product gel extraction x100 dilution PCR product's.

The positive control was unable to be extracted as not enough was loaded.

(17:50 pm) The gel was poured ready for running tomorrow. The samples will need to be Nano dropped before the gel is ran to work out the amount of the marker to load.

(9:00) Luke, Chris, Nathan, Will and Phil are in the lab. Phil is counting up the contents of our swear/latejar to pay into our account later when the bank opens. He will also be decontaminating some things from our area in the class 2 lab. We're just sending/receiving emails at the moment.

(10:00) Luke is about to spec the pentane experiment from last night, as there has been some obvious growth. However after thought it may not be a very conclusive experiment. Will has now combined the tubes and is now doing a nano drop on the gel extraction's from yesterday so we can work out how much to run on the gel. Chris has just came back from getting the shoes Ellen from the environment team gave us as a Citizen Science experiment for phil to analyse and is now working out the amounts of extract to use on the gel-ception gel Lane organisation and volumes
x
x
4μL of DNA Hyperladder ( 600bp band = 48ng 400bp band = 32ng)
2μL of DNA from extract labelled 4 ( according to the Nano 32.8ng)
2μL of DNA from extract labelled A ( according to the Nano 24.6ng)
2μL of DNA Hyperladder ( 600bp band = 24ng 400bp band = 16ng )
2μL of DNA from extract labelled 8 ( according to the Nano 22.4ng)
2μL of DNA from extract labelled 10 ( according to the Nano 22.8ng)
2μL of DNA Hyperladder ( 600bp band = 24ng 400bp band = 16ng )
2μL of DNA from extract labelled 5 ( according to the Nano 21.0ng)
2μL of DNA from extract labelled 1 ( according to the Nano 17.4ng)
1ul of DNA Hyperladder ( 600bp band = 12ng 400bp band = 8ng )
x
x

(11:00) Luke has just finished specing the overnight broth cultures, though the results are inconclusive. We realised that we should have inoculated with a set amount of bacteria in solution, rather than just a swab (like we used). We will set up a rerun this afternoon so that we can analyse whether the bacteria use polystyrene or pentane in preference.

(11:20) Chris has just loaded the gel and it is running at 120V.

(11:45) Chris and Nathan head to the coffee room to discuss future ideas for the project as well as analysis on what we're currently doing.

(13:30) Luke is just starting the pentane experiment again (see yesterday). The protocol is virtually the same, though this time, he's using 0.1 ml less minimal media in the tubes needing the yellow bacteria, and replacing it with 0.1 ml of a 1ml yellow colony bacteria suspension in phosphate buffer solution (PBS)(so as to make the amounts of bacteria in the broths more constant to reduce error significantly). This could take a while. Chris stopped the gel and went down to the transilluminator:

As poor amounts of DNA have been successfully extracted so far, Chris is now troubleshooting the gel extraction kit. unfortunately it also means we have to gather and PCR more DNA again.

(15:00) Luke has just finished preparing the pentane experiment. We're hoping by reducing the errors in bacterial concentration, a pattern will be made clearer. The tubes used have just been put on a shaker, and started off at 240rpm. It will be sampled tomorrow morning, and then Friday morning to determine the growth. Chris has now found the 1/100 dilutions of the last gel extraction which we amplified the DNA of, however Tony and Phil are going to do the Boilate to obtain DNA and run this in the PCR as well.
Nathan and Will start off the triplicate experiment, first by making up a 1L minimal media mixture.

(15:30) to test the Gel extraction kit, Dr Badge supplied 1kb DNA ladder of known DNA concentration to pass through the coulomb to see what % recovery there is, or if there is no DNA recovery in the final aliquot as with the other gel extractions. This time using the new reagents from the box rather than the ones we have currently been using to see if this is the problem fingers crossed the new reagents will be better. if not, we will have to use the second gel extraction kit that we have.
With the minimal media prepared it is set up with 10 ml amounts in 20 corning tubes and 0.5g of polystyrene "sugar" is set up in the necessary tubes, and for the rest of the working day Will and Nathan work on inoculating each of the samples into their respective tubes, in preparation for further testing after staying in the shaker for enough time.

(16:00) Luke has gone to the coffee room to work on degenerate primers for the rest of the afternoon- its surprisingly difficult building in the degeneracy, as he's trying to build in as many alternative codons as possible (the reverse primers are the hardest).

(16:30) Chris has now finished the gel extraction and has put the tubes in the fridge ready to run in the morning . Chris is now setting up the master mix for the PCR in the PCR hood as we are doing 13 PCR reactions, we need to make up 270ul of the master mix which is enough for aliquots of 18µl in 15 reactions. Reaction mixture list:
171µl PCR H₂0
60µl HF buffer
6µl dNTPs
15µl Primer A 28f AAGAGTTTGATCCTGGCTCAGA
15µl Primer B 519R GWATTACCGCGGCKGCTG
2µl Template DNA to be added later to each PCR tube as it is not PCR clean
3µl DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18µl aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive P. aeruginosa DNA and negative bench H₂O controls all samples were added at 2µl to make a final volume of 20µl.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)

DNA Samples: ( use this for loading of the gel)
1 = +ve control Aeruginosa
2 = 100x dilution of orange gel extract ( from previous)
3 = 100x dilution of mixed gel extract ( from previous)
4 = 100x dilution of yellow gel extract ( from previous)
5 = Yellow boilate
6 = Yellow boilate
7 = Yellow boilate
8 = Yellow boilate
9 = orange boilate
10 = orange boilate
11 = orange boilate
12 -ve control hood PCR water
13 -ve control bench H2O

(9:00) Will is making a gel ready to load the PCR reactions and the gel extraction test while Chris and Luke are writing up the wiki for yesterday.

(10:00) Luke has just made up and poured 2 gels ready for electrophoresis. Nathan removes the tubes from the PCR machine and loads them with dye, ready for gel electrophoresis.

(10:30) Phil loads the samples into a gel. This is the PCR which Chris did the previous night. Gel lane organisation:
1 = +ve control aeruginosa
2 = 100x dilution of orange gel extract (from previous)
3 = 100x dilution of mixed gel extract (from previous)
4 = 100x dilution of yellow gel extract (from previous)
5 = Yellow boilate
6 = Yellow boilate
7 = Yellow boilate
8 = Yellow boilate
9 = Orange boilate
10 = Orange boilate
11 = Orange boilate
12 = Marker Hyperladder 5μl
13 = -ve control hood PCR water
14 = -ve control bench H₂O
Each lane was loaded with 20μl of sample as this was the most you could accurately load.

(11:00) Chris is preparing the primers and showing Luke how to prepare the primers made up earlier today, ready for PCRing tonight. (need detail / protocol)

(13:00) Chris loaded and ran the gel extraction test at 120v on a 2% gel. For this you need to make dilutions of the marker DNA to 100ng/50ng total amount of DNA. Load only 100ng of the samples, taking off 10% of the total volume of the elution from the column.

(14:30) Delivery arrived from NewEngland Biolabs, a new sponsor of some competent cells as well as more DNA ladders etc (need detail). Luke and Phil transilluminate Phil's gel from earlier:

(14:40) Will is preparing the boilate reaction to extract DNA for the PCR later.

(15:00) Nathan is re doing the gel extraction test, while Phil is transilluminating the gel extraction test from earlier (no gel photo). The test recovered too little DNA which is why we are re doing the experiment with new reagents from a new kit. As the PE was a new set it must be the QG at fault. (need detail). Luke is taking 1ml aliquots out of the pentane experiment corning tubes that he set up yesterday. Another 1ml aliquot will be taken tomorrow to compare the differences in growth of the bacteria in different concentrations of pentane, with or without polystyrene.

(15:40) Nathan has almost finished doing the gel extraction test, and Phil is making a new gel ready for it to be run, Will is doing the boilate for the PCR which is to be loaded later, while Dr Badge is setting up the Maxwell prep of the DNA for the PCR as well. Chris is now to work out the reaction mixture for the PCR of the different bacteria with the different primers.

(15:45) Luke and Tony have just finished measuring the absorbances of the aliquots at 600nm of the pentane experiment:
Key:
0.XX pent – volume of pentane added to the minimal broth
Bac - 100µl of bacteria suspended in PBS added to the minimal broth
PS - 0.5g polystyrene ‘sugar’ added to the minimal broth
Abs - absorption at 600nm

Absorbance readings:
0.00 pent, Bac, PS Abs=0.292
0.00 pent, PS Abs=0.014
0.00 pent, Bac Abs=0.255
0.00 pent Abs=-0.009
0.05 pent, Bac, PS Abs=0.317
0.05 pent, PS Abs=0.003
0.05 pent, Bac Abs=0.259
0.05 pent Abs=0.001
0.10 pent, Bac, PS Abs=0.300
0.10 pent, Ps Abs=-0.025
0.10 pent, Bac Abs=0.250
0.10 pent Abs=-0.005
0.20 pent, Bac, PS Abs=0.182
0.20 pent, Bac Abs=0.007
0.20 pent, PS Abs=0.250
0.20 pent Abs=0.001
0.40 pent, Bac, PS Abs=0.203
0.40 pent, Bac Abs=-0.018
0.40 pent, PS Abs=0.260
0.40 pent Abs=0.026

The corning tubes will have another aliquot taken out tomorrow at 15:00, and absorbance will be measured again to determine growth. Hopefully the absorbance will change most in the tubes with polystyrene and bacteria in. Seeing as this is just for the yellow colonies, we will be repeating this experiment twice next week with our orange and mixed colonies.

(11:00) Luke and Will in labs. Luke's just tranilluminated the gel set up last night:

(11:20) Luke's just made up a gel ready for running the PCR from overnight.

(12:00) Luke and Will load up the gel with the PCR from last night, and start it running at 120 volts with the following lane organisation:
1 x
2 5µl DNA HyperLadder
3 P. aeruginosa
4 P. aeruginosa
5 P. putida strain A maxwell prep
6 P. putida strain B maxwell prep
7 Orange colony maxwell prep
8 Yellow colony maxwell prep
9 P. putida strain A boilate
10 P. putida strain B boilate
11 Orange colony boilate
12 Yellow colony boilate
13 -ve control bench
14 -ve control hood

(14:30) Will and Phil stop the gel and transilluminate it. The extractions don't appear to have worked unfortunately.

(15:00) Will and Phil spec the pentane experiment, and get these results: (notation is same as thursday:)
Absorbance readings:
0.00 pent, Bac, PS Abs=0.292
0.00 pent, PS Abs=0.014
0.00 pent, Bac Abs=0.255
0.00 pent Abs=-0.009
0.05 pent, Bac, PS Abs=0.317
0.05 pent, PS Abs=0.003
0.05 pent, Bac Abs=0.259
0.05 pent Abs=0.001
0.10 pent, Bac, PS Abs=0.300
0.10 pent, Ps Abs=-0.025
0.10 pent, Bac Abs=0.250
0.10 pent Abs=-0.005
0.20 pent, Bac, PS Abs=0.182
0.20 pent, Bac Abs=0.007
0.20 pent, PS Abs=0.250
0.20 pent Abs=0.001
0.40 pent, Bac, PS Abs=0.203
0.40 pent, Bac Abs=-0.018
0.40 pent, PS Abs=0.260
0.40 pent Abs=0.026

No entry for this date.

Chris thought up a new way to test if the bacteria are using residual pentane/removing residual pentane from the raw polystyrene sugar to try on Monday. If this works we can confirm that the bacteria are growing with the polystyrene alone.

(8:30) Chris arrives at the lab and prepares the master mix for the PCR. Luke then takes over removing the aliquots when he gets in at 9:00, and the PCR tubes are put in a PCR machine at 10:00.

(9:30) Chris, Will and Nathan are setting up gel extractions using a new kit with a view to extracting the TodX gene later today. Will and Nathan have also made 2 gels.

(10:50) Chris set up aerating polystyrene experiment by putting 20g polystyrene in 500ml duran, putting it in the hybridiser at 60 degrees, to try and vaporise residue pentane in the sugar. Pentane boils at 37 degrees C so this is way above the boiling point so all of the pentane can be said to be removed without expanding, leaving just polystyrene. The hybridiser has no exposed elements so should be safe for the small amount of pentane left to be vaporised. two 10g amounts of poly have also been put into tubes and sealed to act as a time zero. These can then be used in a minimal broth poly orange and yellow growth experiment as we can be sure that there is no pentane left over.

(11:00) Luke has just analysed the pentane experiment results: there is clearly an increase in absorbance by bacteria as pentane concentration also increases. The presence of polystyrene sugar also appears to increase the bacteria's absorbance, indicating an increase in bacteria concentration. However, the bacteria could be growing using residue pentane within the sugar.

(13:00) gel extractions are now ready to be ran after nano dropping, and the PCR has finished, Chris changed the program for this run as there was a lot of other interference bands of other amplified areas of DNA with some very large... to combat this the extension time has been reduced to one minute to only give enough time to amplify the shorter bands
PCR cycle program used was iGEMTOL2 which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
65 degrees C - 30 seconds
72 degrees C - 1 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)

(12:00) Luke is designing new primers for the genes we've managed to extract, that incorporate the biobrick prefix/suffixes.

(1:15) will did nano drop of gel extraction using a different kit- zymo.

(14:10) Chris has prepared and loaded the TobB and the TodF gels and has set them running at 120V for 2.5 hours, the gel extraction nano drop results had a lot of salt contamination, so we are now looking into trouble shooting this, however there was a ok amount of DNA recovery.

(16:20) Chris has finished the gel extraction of the remaining samples from the TodX gel putting through 2x 200μL 1x600μL and 1x400μL as although the protocol said 200μL twice, the bottle of the wash buffer said between 200μL and 600μL. as there was a lot of salt the extra washes should remove this.

(17:30) The gel extractions don't appear to have worked:

(9:00) started the day by setting up pcr.

(11:00) Chris prepared and loaded the PCR gel of the 16S samples from last night, running at 120V on a 2% gel for 2.5 hours lane organisation:
+ve Aeruginosa
orange boilate
orange boilate
orange boilate
yellow boilate
yellow boilate
yellow boilate
marker 5μL ( new England biolabs 100bp )
-ve bench
-ve hood

(12:00) Chris prepared and loaded the gel extractions, earlier on he ran though another 10μL of distilled water through the coulomb to see if there was any DNA left, so there are two lots of gel extractions being run. gel was ran at 120V on a 2% gel lane organisation:
4μL marker (New england Biolabs 100bp)
orange boilate (9) extraction
putida maxwell A(6B) extraction
putida Maxwell A(6A) extraction
4μL marker (New england Biolabs 100bp)
putida Maxwell B(5B) extraction
putida Maxwell B(5A) extraction
orange boilate (9) extraction number 2
putida maxwell A(6B) extraction number 2
putida Maxwell A(6A) extraction number 2
2μL marker (New england Biolabs 100bp)
putida Maxwell B(5A) extraction number 2
putida Maxwell B(5B) extraction number 2
1μL marker (New england Biolabs 100bp)

(13:40) Chris Stopped the PCR gel and the gel extraction gel:

(14:00) From this Chris is now setting up the samples for a sequencing reaction Dr Badge is going to run over night, only using the reverse primer. primer is diluted three fold from the 10μM concentration, and 2μL of each of the 5A and 5B are to b sequenced as these were the best extracts.

(14:30) after analysis of the 16S PCR from last night, there is contamination in the -ve controls, both bench and hood... this was using new polymerase, DNTP's and buffers so the only suspects for the contamination are the primers and PCR water. As we have ran out of PCR water this is going to be fresh for the next set of reactions, however the todC1 used the same water so we can compare when this is run on the gel.

(15:00) Chris is now preparing the todX PCR samples from this morning with the old polymerase (had a slight problem so the todC1 and todG PCR reactions used fresh Polymerase) this can test the polymerase for contamination from the 16s experiment. loading 5μL of loading dye into each sample. Luke and Chris are making two new gel's ready for these to be run, with the 3rd gel in the gel tank.

(16:20) Tony has now loaded the gel for the TodX PCR from this morning running it at 120V Lane organisation:
1 P. aeruginosa
2 P. aeruginosa
3 P. putida strain A maxwell prep
4 P. putida strain B maxwell prep
5 Orange colony maxwell prep
6 Yellow colony maxwell prep
7 P. putida strain A boilate
8 P. putida strain B boilate
9 Orange colony boilate
10 Yellow colony boilate
5μL marker (New england Biolabs 100bp)
11 -ve control bench
12 -ve control hood
Chris and Luke are editing the Wiki while the gel's are cooling.

(17:00) Chris is now setting up the Re amplification on the TodX Gel extraction, as there are only 5 samples the PCR has only been set up for 10 to save on reagents, only doing each one once and the controls. Chris is now setting up the master mix for the PCR in the PCR hood as we are doing 9 PCR reactions, we need to make up 180ul of the master mix which is enough for aliquots of 18ul in 10 reactions. Reaction mixture list:
112ul PCR H20
40ul HF buffer
4ul dNTPs
10ul Primer A TODXF 5'-atgcccgccagtctgacgcttg-3'
10ul Primer B TODXR 5'-accagccagcaccatgcggc-3'
Template DNA to be added later as it is not PCR clean
2ul DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive P. aeruginosa DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul.
PCR cycle program used was iGEMTOL which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 1 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)
the part in italics is the cycle which is repeated 30 times.

(17:40) Chris just loaded the PCR and is now preparing the samples to be ran over night on the gel at 20V. As there will be a lot of gels to do in the morning, we're thinking this is the best use of time. Luke is editing some of the modelling page and italicising bacteria names on the Wiki while Tony is re streaking the orange and yellow colonies. Lane organisation was the same for both todC1 and todG :
x
1 P. aeruginosa
2 P. aeruginosa
3 P. putida strain A maxwell prep
4 P. putida strain B maxwell prep
5 Orange colony maxwell prep
6 Yellow colony maxwell prep
7 P. putida strain A boilate
8 P. putida strain B boilate
9 Orange colony boilate
10 Yellow colony boilate
5µl DNA HyperLadder
11 -ve control bench
12 -ve control hood

(17:55) Luke has just stopped and transilluminated the gel ready for us to go home:

Chris has just finished loading the gel and has set it running at 20 volts.

(9:00) Chris came in and stopped the gel's with Luke and Nathan close behind. After transilluminating, Chris realised that the power pack was set too high (120v instead of 20v), so all of the DNA had run off the end of the gel:

This means that once the PCR has been stopped we will need to re-run these primers to re extract the DNA from yesterday. As all of the DNA had been run off the gel, Nathan is now remelting the gel so we can reuse it to save on costs, ready to run some of the TodX Gel extraction PCR amplification from last night's PCR. as this time the reaction should have only been able to amplify single bands, we are going to do a PCR purification rather than run it all and gel extract due to the low yields.

(10:15) Nathan has just finished making the first gel after a few problems with the melting. Luke is updating the modelling page and Chris is about to set up the PCR for the TodC1 and TodG samples. hopefully by the time these are ready we can then run 2µl of last nights PCR to see what bands are present.

(10:30) Chris is now setting up the master mix for the PCR of the TodG and TodC1 in the PCR hood as we are doing 12 PCR reactions, we need to make up 270ul of the master mix which is enough for aliquots of 18ul in 15 reactions. Reaction mixture list:
171ul PCR H20
60ul HF buffer
6ul dNTPs
15ul Primer A
15ul Primer B
Template DNA to be added later as it is not PCR clean
3ul DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive P. aeruginosa DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 1 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)
the text in italics is the cycle, which is repeated 30 times.

(11:30) Chris is now putting the DNA into the PCR reactions ready to run the PCR. Nathan is re making one of the gel's and Luke is editing the modelling page. Had a slight problem with the gel with a spillage so Chris and Sue are clearing that up making sure there is no ETBR on the surfaces. Luke is now making some more gel to replace the spilt stuff.

(12:00) PCR is now running and Chris is preparing the samples for running some of the TodX gel extraction re amplification gel as we are going to do PCR purification rather than gel extraction, only 2μL of the 5 reactions are to be loaded so we have 18 micro litres DNA to purify . Samples were made by adding 4μL of loading dye to 14μL of TE buffer and 2μL of each of the sample. the aeruginosa and -ve control's had 5μL of loading dye added and were loaded completly as they were more to test the PCR rather than for extraction of the DNA.

(12:40) Chris has now loaded the gel and is running it at 120V, Lane organisation:
x
-ve aeruginosa
-ve aeruginosa
5μL marker (New england Biolabs 100bp)
number 6A
number 6B
number 5A
number 5b
number 9
5μL marker (New england Biolabs 100bp)
-ve hood
-ve bench
x
x

(14:00) Gel has been transilluminated:

Chris is now going to do a PCR purification of the 18micro litres of the PCR left. fingers crossed this works well!

(15:00) Nathan is now preparing the samples of the PCR Chris set up this morning ready for loading while Luke is looking over the enzymes we need for the insertion of the DNA into the biobrick. PCR purification is done and Chris is getting it Nano dropped with Colin downstairs, Data to be added here soon, Looks good though! max of 47ng/ul min of 32ng/ul for the 5 samples so great results for the TodX PCR purification (QIAGEN).

(15:50) Chris and Luke have now loaded the samples on to the gel's and set them running at 120V until the end of the day and is now discussing the ligation with Dr Badge as well as trying to get some more 16S primers so we can do some more PCR on the orange and yellow bacteria. Lane organisation: (same for both gel's)
1 P. aeruginosa
2 P. aeruginosa
3 P. putida strain A maxwell prep
4 P. putida strain B maxwell prep
5 Orange colony maxwell prep
6 Yellow colony maxwell prep
7 P. putida strain A boilate
8 P. putida strain B boilate
9 Orange colony boilate
10 Yellow colony boilate
5µl marker (New england Biolabs 100bp)
11 -ve control bench
12 -ve control hood

(17:16) Nathan and Luke are now doing the spec 600nm on the airated Poly experiment while chris is writing the wiki.

(17:30) Chris is now setting up the master mix for the PCR 16S orange and yellow as we have been donated more universal 16S primers from Dr Richard Haigh. This is done in the PCR hood as we are doing 9 PCR reactions, we need to make up 270ul of the master mix which is enough for aliquots of 18ul in 15 reactions. Reaction mixture list:
112ul PCR H20
40ul HF buffer
4ul dNTPs
10ul Primer A
10ul Primer B
Template DNA to be added later as it is not PCR clean
2ul DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive P. aeruginosa DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)
the text in italics is the cycle, which is repeated 30 times

(17:50) After the PCR was set up, Chris and Nathan swiftly stopped the gel's and got them on the transilluminator. the results from the PCR of the todC1 and TodG look promising for the C1, however again there is two bands at about the correct position.

(9:00) in and straight away making gel's , Chris is sterilising the bottles while Nathan and Will are getting ready to make the gel. Luke is creating a task list.

(10:00) Luke is re formatting photos ready to go on the Wiki and Will is doing the Boilate as from looking at last nights gel's the boilate DNA hasn't amplified any bands... Tony and Nathan are pouring the gel's ready to be loaded while Chris is analysing the gel photos of the tod genes.

(10:30) After the pc crashed... losing lots of wiki work, Chris and Nathan are now re-writing the entrance for today.

TodC1 - 1353bp
> Aeruginosa - band at 800bp and 1kb
> Maxwell reaction
> Putida A and b - Band at 800bp, 1.2kb and one in-between 1.2 and 1.5kb
> Orange bacteria - one band high up above the 1.5kb marker
> yellow bacteria - no bands
> Boilate reaction
> boilates failed to amplify

TodG - 807bp
> Aeruginosa - band at 1kb and one high up
> Maxwell reaction
> putida A and b - band at 700bp and a fainter one at 800 and 600 ... bands also present above the 1.5kb marker
> Orange good sized band at 800bp and a lower one at 700bp ... also has bands at 1.2 and 1.5 kb
> yellow faint band at 800bp and one at 1kb
> Boilate reaction
> boilate failed

TodX - 544bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - Bands at <500bp, 600bp, 1200bp and >1500bp
> Orange bacteria - 1 band at >1500bp
> Yellow bacteria - Faint bands at 1200bp and >1500bp
> Boilate reaction
> Putida A and B - Faint bands at <500bp, 600bp, 1200bp and >1500bp
> Orange and yellow boilates failed.

TodB - 324bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - None
> Orange bacteria - several non distinct bands ranging from >300bp to 800bp, 2 are between 300bp-400bp
> Yellow bacteria - several non distinct bands ranging from >200bp to 1500bp, 1 faint band is between 300bp-400bp
> Boilate reaction
> Putida A and B - No distinct bands
> Orange - one faint band between 300bp-400bp and more faint bands between 400bp-1000bp
> Yellow - None

> TodF - 460bp
> Aeruginosa - None
> Maxwell reaction
> Putida A and B - 1 band between 700bp-800bp
> Orange bacteria - 1 band at 1500bp and 1 band between 1200-1500bp
> Yellow bacteria - Faint band between 500bp-600bp, other bands are above
> Boilate reaction
> Putida A and B - Faint band between 500bp-600bp, other bands are above, 1 strong band between 700bp-800bp
> Orange - Strong bands between 1000bp-1500bp
> Yellow - Faint band between 500bp-600bp, and strong bands >1200bp

(11:00) As the gel is set, Chris is now loading the 16s gel and the test of the PCR purification, loading (10%) 3µL of each of the samples with 4µL of loading dye and 13µL of TE buffer to make each sample for loading. gel is running at 120V for 2 hours. lane organisation:
5µL 100bp Marker, ( New England Biolabs)
6a
6b
5a
5b
9
1µL 100bp marker ( New England Biolabs)
10µL 100bp marker ( New England Biolabs)
the different amounts of DNA ladder are to see if the concentrations from the Nano Drop are correct.

(11:40) Chris stopped and transilluminated the 16S gel after it only being ran for a hour, surprisingly there are bands for all of the reactions, however, there are also bands in the -ve bench and a lighter band in the -ve hood lane, as these were fresh primers, DNTp's, there was no contamination last time we used the pol and reaction buffer... the only thing it can be is the PCR H2O or technique.. so we will need to obtain a fresh stock of PCR H2O for the next set of reactions. unfortunately this sets us back again, and as the primers have also yet to arrive the day has yet to kick start.

(13:30) Chris Stopped the Gel Purification gel which Luke then transilluminated. There is DNA in the purification and it is of high ng/ul which is good. Unfortunately however we have just got the sequence data back from the DNA and it looks like we haven't extracted the TodX gene itself, which is disappointing. As before we only sequenced from the reverse primer, we are to re do this with the forward primer to get the full set of data. this calls however for a meeting

(14:30) Chris is preparing for PCR for the 16s , todX and TodF as the TodF PCR gel has some bands which look promising. At the same time Chris is running the original PCR of the TodF gel, at 150V transilluminating every 15 min.

(15:00) will has now taken over the running of the PCR gel, the bands are still very hard to see, however don't seem to be improving. Chris is now setting up the master mix for the PCR 16S orange and yellow This is done in the PCR hood as we are doing 9 PCR reactions, we need to make up 180ul of the master mix which is enough for aliquots of 18ul in 10 reactions. Reaction mixture list:
112ul PCR H20
40ul HF buffer
4ul dNTPs
10ul Primer A
10ul Primer B
Template DNA to be added later as it is not PCR clean
2ul DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where the DNA was added, and the positive P. aeruginosa DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)
the text in italics is the cycle, which is repeated 30 times
as well as that Chris is setting up the TodX and todF which PCR under different conditions and as there are 12 reactions, the mixture is for 15. Program used was iGEMTOL2 - used previously.

(15:50) Nathan and Tony are doing spectrophotometry on the aerated Poly experiment - results to follow while Luke has almost finished putting the photos into the wiki.

(16:30) Chris is now analysing the gel photos from the todF re run gel in a hope to find the bands again, after looking at the gel under the cutting UV light, no bands can been seen to actually cut them out of the gel... Luckily the PCR from today will give us more DNA. so to not waste more time, we will Freeze thaw the bands, to extract some DNA to amplify from the band directly to save on time, as well as doing the Zymogen gel extraction.

(17:30) preparing for tomorrow by making gel's and Wiki'ing. After labs today, Chris and Luke are going to go to Asda to get preparations for the cake sale on saturday at the open day.

(8:50) Chris is preparing the samples for the 16S PCR to be ran on a gel, while will is making two new gels ready to run the tod PCR's. Bad news from the Asda trip, so will have to pop to Morrison's after labs.

(9:30) Gel running with the following organisation: Chris, insert lanes here

Luke and Nathan just got in and are getting ready for the day ahead.

(10:00) Will is making and pouring a couple of gels for the TodX and TodF PCR gel electrophoresis.

(10:20) Luke is now preparing the samples of the todX and todF PCR reactions from last night for running on the gel, while Chris is writing the Wiki for yesterday/this morning so far.

(11:00) Luke has just set up the next gels, using the TodF and TodX PCRed out last night. The lane organisation is:
1 x
2 P. aeruginosa
3 P. aeruginosa
4 P. putida strain A Maxwell prep
5 P. putida strain B Maxwell prep
6 Orange bacteria Maxwell prep
7 Yellow bacteria Maxwell prep
8 P. putida strain A boilate
9 P. putida strain B boilate
10 Orange boilate
11 Yellow boilate
12 5µl 100bp DNA ladder
13 bench H₂O
14 hood H₂O

(11:30) Luke has just transilluminated the 16S gel from 9:30:

(13:30) Luke has just transilluminated the TodX gel:

The TodF was on a longer gel, so is not ready to transilluminate yet.

(14:00) Luke transilluminated the TodF gel- it doesn't look like we have any of the gene we targeted in any of the DNA samples.

(15:00) Chris is starting to gel extract from the PCR gels ran this morning/early afternoon.

(16:00) Luke is currently tidying up the lab a bit.

(17:00) Gel extract didn't go very well, so Luke and Chris are now preparing the PCR. Luke is now setting up the master mix for the PCR 16S orange and yellow This is done in the PCR hood as we are doing 9 PCR reactions, we need to make up 180ul of the master mix which is enough for aliquots of 18ul in 10 reactions. Reaction mixture list:
112ul PCR H20
40ul HF buffer
4ul dNTPs
10ul Primer A
10ul Primer B
Template DNA to be added later as it is not PCR clean
2ul DNA Polymerase
These reagents were added then as the DNApol was in glycerol the tube was mixed and spun for a second to remove bubbles. dNTPs cannot be freeze thawed so whatever was left was thrown. After this, Chris put 18ul aliquots of the master mix into 9 separate PCR eppendorfs and prepared the negative control while in the PCR hood with the PCR clean H2O. The tubes were then taken to the bench where Chris added the DNA, and the positive P. aeruginosa DNA and negative bench H2O controls all samples were added at 2ul to make a final volume of 20ul. Chris then placed the tubes into the PCR block and set it going.
PCR cycle program used was iGEM16S which is as follows:
98 degrees C - 5 min
98 degrees C - 30 seconds
50 degrees C - 30 seconds
72 degrees C - 2 minutes
72 degrees C - 5 minutes
15 degrees C - Forever ( this is for the end of the reaction)
the text in italics is the cycle, which is repeated 30 times

University of Leicester open day today, Chris arranged for the team to have a stall outside our building to try and raise some money as well as helping out on the department tours.

No entry for this date.

(9:30) The group met in the computer room to start a day of editing the Wiki, Emily is altering the photos, Luke is doing the project page, will is going through his attributions and putting them into a word doc and Phil is editing the attributions page, while Tony is looking at the poster.

(10:00) 16S PCR has finished so Chris is preparing samples to run on a gel. As there is 20μL 2μL is to be loaded to make calculations easier, the remaining 18μL is to be put through the QIAquick QIAGEN PCR Purification ( Cat. No. 28104). Protocol for the PCR purification can be found on the QIAGEN website, the only alterations to the protocol were step 4 and 7 leaving them for 5 minutes each time before the centrifugation step.

(12:00) Luke poured the gel which Chris has now loaded with the PCR products lane organisation:
Aeruginosa
orange
orange
orange
yellow
yellow
yellow
Marker 5μL (NEB)
-ve hood
-ve bench

(12:40) PCR purification has now been finished and Chris used the nano drop ( After being given permission to do so By Dr Dalgelish ) to work out rough ng/μL for each of the samples. The 260/280 ratios were all around the 1.4 mark with concentrations being between 36 and 46ng/μL for the seven samples. Chris then prepared the samples ready to be run on a gel to work out the concentrations more accurately using 3μL ( 10%) of the 30μL total elution volume making each up to 20μL to be run on the gel. Will made a new 2% gel ready for this to be run after lunch.

(13:00) as the PCR Purification is done, Chris is making up 3.3nM aliquots of the 16S primers ready for the sequencing reaction in the PCR hood, this is done by diluting 10nM primers 1:2 with PCR H2O . Once this had been done Chris gave the samples to Dr Badge ready to be run with forward and reverse primers.

(14:00) Chris stopped and transilluminated the gel, ***insert image*** the bands are in the correct place for the 16S sequences however there is contamination again in the -ve .... hopefully this won't produce to much garbage in the sequencing. Chris then loaded the gel of the PCR Purification products to work out the concentrations, Lane organisation:
x
1μL 100bp Marker (NEB)
4
7
1A
2μL 100bp Marker (NEB)
5
6
4μL 100bp Marker (NEB)
2
3
8μL 100bp Marker ( NEB)

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