Team:NRP-UEA-Norwich/Week7

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=Week 7=
=Week 7=
-
With half the team away for the week, this was a hard week in terms of lab work. The remaining team members, concentrated mainly on doing lab work and in particularly the completion of the growth studies which produced good results which can be analysed. However, there were also many highlights such as the confirmation of our arduously designed gene constructs being made and the arrival of primers allowing us to send our BioBricks to be sequenced.
+
With half the team away for the week, this was a though week in terms of lab work. The remaining team members concentrated mainly on the completion of the growth studies which produced good results that we are looking forward to analysing. However, there were also many highlights such as the confirmation of our arduously designed gene constructs being made and the arrival of primers allowing us to send our BioBricks to be sequenced.
==Day 1 (20/08/12)==
==Day 1 (20/08/12)==
===Research===
===Research===
-
Research was carried out into companies and their requirements for sequencing. This is preparation for sequencing of our own BioBricks (B-M and M-B in pSB1C3. Sequencing is required to confirm they are the correct sequences before we can send them to iGEM. Lukas found that the requirements for Bio-LifeSciences was 5-6µL of plasmid DNA at a concentration of 100ng/µL and the primers of quantity of 10µL and concentration of 3.2pmol/µL. The primers were also ordered.
 
-
The genes for synthesis of our comparator circuit DNA constructs were finalised with GenScript. Witt three weeks left we are hoping they will arrive end of week 9 or start of week 10 to get them sent off in time as new BioBricks.
+
Research into companies and their requirements for sequencing was carried out. This is in preparation for sequencing of our own BioBricks (BM and MB in pSB1C3). Sequencing is required to confirm, that our genes have the correct base sequence before we send them to iGEM. Lukas found that the requirements for Bio LifeSciences was 5-6µL of plasmid DNA at a concentration of 100ng/µL and the primers at a quantity of 10µL and concentration of 3.2pmol/µL. The primers were ordered and the plasmid samples aquilotted in preparation.
 +
 
 +
The genes of our comparator circuit DNA constructs were finalised within the requirements of GenScript. With three weeks left, we are hoping they will arrive at the end of week 9 or the start of week 10. This will hopefully give us enough time to get into the BioBrick format, send them off to iGEM and characterise and prove their function.
===Labs===
===Labs===
-
As the previous transformations of the BioBricks BBa_E0420 (CFP) and BBa_K081014 (RFP) were either unsuccessful or showed contamination, we decided to retry the transformation with a different team member. The procedure used was that seen on the lab protocol page. In addition, a further BioBrick was transformed: BBa_K206009 (GFP). Again, this will also be used in the characterisation of our BioBricks as these will eventually be ligated to our promoter. The transformants of these were plated on ampicillin plates of a concentration of 100µg/ml. A negative control of E.coli cells were plated also on ampicillin plates.
+
As the previous transformations of the BioBricks BBa_E0420 (CFP) and BBa_K081014 (RFP) were either unsuccessful or showed contamination so we decided to retry the transformation with a different team member. The procedure used was the one seen on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol lab protocol page]. In addition, the BBa_K206009 (GFP) BioBrick was transformed. Again, this will also be utilised in characterisation of our BioBricks as these will eventually be ligated to our promoter. The transformants were plated on ampicillin plates of a concentration of 100µg/ml. A negative control of ''E.coli'' cells was implemented as well.
-
 
+
-
We also made a plan of requirements for sequencing of B-M and M-B. We have already planned for the requirements but here we set aside the DNAs we needed and pipetted them into separate eppendorfs.
+
-
 
+
==Day 2 (21/08/12)==
==Day 2 (21/08/12)==
Line 25: Line 23:
===Labs===
===Labs===
-
A message from GenScript confirming synthesising of ordered genes!
+
A message from GenScript confirming the synthesising of the ordered genes!
-
The transformation was unsuccessful, there was no sign of contamination as there were no growth on any of the plates. A transformation of CFP, RFP and GFP into alpha competent cells was repeated. Some changes were made: positive controls were made (plasmids containing M-B with backbones that contain ampicillin resistance and chloramphenicol resistance). Through some slight confusion, GFP had been plated on ampicillin plates the previous day, as the BioBrick contains only chloramphenicol resistance, it was plated on chloramphenicol plates (25µg/ml). The rest of the protocol was unchanged from the previous day. Some of the transformation culture was retained and more LB medium was added and grown overnight.
+
The transformation was unsuccessful as there was no sign of contamination and no growth showed on any of the plates. A transformation of CFP, RFP and GFP into alpha competent cells was repeated. Some changes were made: positive controls were made (plasmids containing MB within backbones that contain both ampicillin and chloramphenicol resistance). Through some slight confusion, GFP had been plated on ampicillin plates the previous day, as the BioBrick contains only chloramphenicol resistance, it was plated on chloramphenicol plates (25µg/ml) today. The rest of the protocol was unchanged from the previous day. Some of the transformation culture was retained and more LB medium was added and incubated over night.
-
A growth study involving PyeaR-GFP (BBa_K381001) BioBrick and alpha cells was planned again but this time involving M-B and B-M BioBricks too. The plan is grow these cells in LB medium without antibiotic resistance in sterile conditions overnight to increase the number of cells. The next day, these will be pelleted down to obtain a large number of cells and these will be diluted down with LB both till an absorbance of 0.2 is reached. From then on, these will be run for 12 hours.  
+
A growth study involving the PyeaR-GFP (BBa_K381001) BioBrick and alpha cells was planned again but this time involving MB and BM BioBricks as well. We hope to obtain results that will allow us to compare the data sets. The plan is to grow these cells in LB medium without antibiotic resistance in sterile conditions overnight to increase the number of cells. The next day, these will be pelleted down to obtain a larger cell concentration and these samples will be diluted down with LB both till an absorbance of 0.2 is reached. From then on, measurements will be run for 12 hours ([https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_comparison_between_the_growth_of_E.coli_cells.2C_before_and_after_transformation_with_PyeaR_.2B_GFP_.28BBa_K381001.29_and_B-M_and_M-B_.28in_pSB1C3.29-_Lag_Phase_Study Results]).
==Day 3 (22/08/12)==
==Day 3 (22/08/12)==
Line 37: Line 35:
[[File:BM-MB S+P.png | thumb | '' '''Figure 1.''' B-M/M-B in pSB1C3 plasmid preps, double digested using PstI and SpeI to linearize for later ligation with fluorescent reporter'']]
[[File:BM-MB S+P.png | thumb | '' '''Figure 1.''' B-M/M-B in pSB1C3 plasmid preps, double digested using PstI and SpeI to linearize for later ligation with fluorescent reporter'']]
-
Following the plan of the growth study plan set up the previous day, Khadija, Lukas and Rebecca arrived into the labs bright and early and began the growth study. The full experiment details can be found on the experiments page (including statistical analysis and graphical data).
+
Following the protocol of the growth study set up the previous day, Khadija, Lukas and Rebecca arrived into the labs bright and early. The full experiment details can be found on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments Experiments page].
-
The transformation concerning the GFP, RFP and CFP were successful. There was growth on all the plates except from the negative control plates. Some plates showed greater numbers of colonies than others. The positive control plates showed lots of colonies. The GFP plates showed lots of growth, too. The RFP and CFP showed less growth. There were less than 10 colonies on these plates. Inoculations of these were made into LB media and incubated overnight at 37 degrees Celsius.  
+
The transformations of the GFP, RFP and CFP were successful. Growth was observed on all plates except the negative control plates. Some showed greater numbers of colonies than others. The positive control showed lots of viable cells. The GFP plates showed lots of growth, too. The RFP and CFP showed less growth. There were less than 10 colonies on these plates. Inoculations with viable cells from all samples were made into LB media and incubated overnight at 37°C.  
-
A restriction digest was carried out by Khadija to both validate and linearise B-M and M-B within the iGEM pSB1C3 backbone. It was linearised in preparation of ligating a reporter to the insert (B-M and M-B hybrid promoter). The restriction digest was carried out using ''Pst''1 and ''Spe''1. This was left overnight to fully complete the digestion. From previous experiments which ligated M-B and B-M into pSB1C3, this produced 4 B-M's and 4 B-Ms labeled BM1-4 and M-B1-4. These were digested (''Figure 1.'').
+
A restriction digest was carried out by Khadija to both validate and linearise BM and MB within the iGEM pSB1C3 backbone. It was linearised in preparation of ligating a reporter to the insert (B-M and M-B hybrid promoter). The restriction digest was carried out using PstI and SpeI. The samples were left overnight to fully complete the digestion. From previous experiments which ligated MB and BM into pSB1C3, we used 4 BM's and 4 BMs samples labeled BM1-4 and M-B1-4 respectively. These were used in the digestion (''Figure 1.'').
-
The running inoculations of M-B and B-M BioBricks were miniprepped following the protocols on the lab protocol page. This produced more plasmid DNA. These need to be nanodropped.
+
The running inoculations of MB and BM BioBricks were miniprepped, following the protocols on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol lab protocol page]. This produced more plasmid DNA which was nano dropped.
==Day 4 (23/08/12)==
==Day 4 (23/08/12)==
Line 49: Line 47:
===Labs===
===Labs===
-
The overnight restriction digest was run on an agarose gel (1% w/v) for an hour and a half. The image showed that MB1, MB2, MB4 and BM2 were fully cut. These showed only one band. The small fragment which would be only a few base pairs long was not seen as expected. Some of the samples ran further, we expect that these are uncut plasmids (''Figure 1.'')  
+
The overnight restriction digest was run on an agarose gel (1% w/v) for an hour and a half. The image showed that MB1, MB2, MB4 and BM2 were fully cut and presented with only one band. The smaller excised fragment, which would be only a few base pairs long, was not visible as expected. Some of the samples ran further than exspected and we hypothesised that these might be uncut plasmids (''Figure 1.'').
-
BioBrick (BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP)) transformants that had been grown in culture were miniprepped (plasmid isolation protocol in lab protocol) by Lukas to attain pure plasmid DNA. This was stored in the freezer.
+
BioBrick (BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP) transformants were miniprepped by Lukas to attain pure plasmid DNA. These was stored in the freezer.
-
As the Growth study carried out the previous day did not show the lag phase as the starting concentrations were too high, a lag phase only study was carried out. This involved starting the cultures at a lower absorbance and hence concentration. The absorbances started at were around the 0.04 marker however, dilution of these to reach an exact 0.04 was very difficult as there were many errors in the machine, therefore, 10µl of cultures grown overnight were pipetted into 1ml of LB broth media in a cuvette. The study lasted for 5 hours with readings taken every hour.  
+
The growth study carried out the previous day did not include the lag phase as the starting concentrations of cells were too high. A lag phase only study was carried out. This involved starting measurements at a lower cell concentration (relative to absorbance). The absorbances starting point was chosen at around 0.04, however reaching these values precisely was very difficult due to the error margin of the spectrophotometer being great in this range. Therefore 10µl of cultures grown overnight were pipetted into 1ml of LB broth media in a cuvette. The study lasted for 5 hours with readings taken every hour. Results are shown on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments#A_comparison_between_the_growth_of_E.coli_cells.2C_before_and_after_transformation_with_PyeaR_.2B_GFP_.28BBa_K381001.29_and_B-M_and_M-B_.28in_pSB1C3.29-_Lag_Phase_Study experiments page].
-
The primers ordered arrived! iGEM sequencing primer (VF and VR) stock was made up to 100µM by resuspension in water. These were diluted to match the requirements of Bioscience of 3.2pml/µL. This is sufficient for 10 reactions. 3.2µL of this was further diluted with 96.8µL distilled water to get 100µL of primer at a concentration of 3.2pml/µL. This is sufficient for 10 reactions. Following the arrival and preparation of primers, the plasmid DNA of our BioBricks to be sent for sequencing were prepared. The made DNA was packaged into separate eppendorfs to be sent off. Each eppendorf contained 6µL of the DNA (MB1-4 and BM(1-4) and was labelled. As there was a delay, these were unable to be sent off immediately and were frozen again until possible to be sent off.
+
The sequencing primers have arrived! iGEM sequencing primer (VF and VR) stock was made up to a concentration of 100µM by resuspension the stock solution in distilled water. These aquilots were diluted to match the concentration requirements of Bioscience at 3.2pml/µL. Following the arrival and preparation of primers, the plasmid DNA of our BioBricks to be sent for sequencing was prepared. The made DNA was packaged into separate eppendorfs to be sent off. Each eppendorf contained 6µL of the DNA (MB1-4 and BM(1-4) and was labelled accordingly. As there was a delay, these were unable to be sent off immediately and were frozen again until postage.
==Day 5 (24/08/12)==
==Day 5 (24/08/12)==
===Labs===
===Labs===
-
[[File:28.png | thumb | '' '''Figure 2.''' Gel electrophoresis of Isolated plasmids of GFP, CFP, RFP Biobricks, single digested with PstI'']]
+
[[File:28.png | thumb | '' '''Figure 2.''' Gel electrophoresis of Isolated plasmids of GFP, CFP, RFP BioBricks, single digested with PstI'']]
-
Rachel carried out a gel purification (on both MB and BM from the gel run) using Promega Wizard kit (Ref: A9281) as seen in the lab protocol page. From the gel, the lanes containing BM1, BM2, BM3, BM4, MB1, MB2 and MB4. These refer to bacterial mammalian and the mammalian bacterial promoter transformed cells from different colonies. The purified samples were stored in the freezer.
+
Rachel carried out a gel purification (on both MB and BM fragments) using a Promega Wizard kit (Ref: A9281) as described in the [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol lab protocol page]. The purified samples were stored in the freezer.
-
Rebecca transformed BioBrick BBa_K561002 (PFDHF +RFP + TETR) into Bioline Alpha Select Gold Standard cells following the transformation protocol as seen in the lab protocols page. The negative control was simply alpha cells, untransformed. The transformation was plated onto agar plates containing chloramphenicol resistance at 25µg/ml.
+
Rebecca transformed BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K561002 BBa_K561002] (PFDHF +RFP + TETR) into Bioline Alpha Select Gold Standard cells following the transformation protocol as seen on the [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol lab protocol page]. The negative control was simple, untransformed alpha cells. The samples were plated onto agar plates containing chloramphenicol at 25µg/ml.
-
Khadija nanodropped the miniprepped plasmid BioBricks BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP). As many cultures were grown per a single colony on the plates, there were separate epppendorfs containing the plasmid DNA. In total there were 4 of CFP, RFP and GFP samples. These were shown to have high concentrations of DNA ranging from 165ng/µL to 332 ng/µL. Following the nanodrop a restriction digest was carried out to validate the plasmid DNA. Two sets of restriction digests were carried out. One is to cut at ''Pst''1 of the flanking suffix/prefix DNA and the other digest is to cut a specific restriction site in the backbone. As BBa_K206009 is found in pSB1AK3 there is a ''Hind''III site and  BBa_K081014 is in pSB1C3 and contains an ''Eco''RV site. BBa_E0420 contains many restriction sites but none that can be cut by enzymes that we have in immediate stock. Therefore, a second restriction digest was not carried out on this BioBrick. The single digests involved 1µL DNA, 2µL buffer, 0.2µL BSA, 0.5µL of restriction enzyme and 16.3 µL of distilled water. Buffer H was used in the digests using ''Eco''R1, Buffer B for ''Hind''III and Buffer D for ''Eco''RV. These completed digestions were stored in the freezer after 2 and a half hours of digestion ('Figure 2.'')
+
Khadija nanodropped miniprepped plasmid BioBrick samples of BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP). As several morphologically different colonies had grown an several plates, the samples of these cells were stored in labeled epppendorfs. In total there were 4 different plasmid isolations of CFP, RFP and GFP. These were shown to have high concentrations of DNA ranging from 165ng/µL to 332 ng/µL. Following a nanodrop, two restriction digests were carried out to validate the DNA. One cut at the brick insert at the suffix/prefix, the other digested at a specific restriction site within the backbone. As BBa_K206009 is found in pSB1AK3, there is a HinIII site and  BBa_K081014 is in pSB1C3 and contains an EcoRV site. BBa_E0420 contains many restriction sites, but none that can be cut by enzymes that we had in stock. Therefore, a second validation restriction digest was not carried out on this BioBrick.
 +
The protocol involved 1µL DNA, 2µL buffer, 0.2µL BSA, 0.5µL of restriction enzyme and 16.3 µL of distilled water. Buffer H was used in the digests with EcoR1, Buffer B for HindIII and Buffer D for EcoRV. These completed digestions were stored in the freezer after 2 and a half hours of digestion ('Figure 2.'')

Latest revision as of 00:35, 27 September 2012

Header1NewGreen.png

NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Contents

Week 7

With half the team away for the week, this was a though week in terms of lab work. The remaining team members concentrated mainly on the completion of the growth studies which produced good results that we are looking forward to analysing. However, there were also many highlights such as the confirmation of our arduously designed gene constructs being made and the arrival of primers allowing us to send our BioBricks to be sequenced.

Day 1 (20/08/12)

Research

Research into companies and their requirements for sequencing was carried out. This is in preparation for sequencing of our own BioBricks (BM and MB in pSB1C3). Sequencing is required to confirm, that our genes have the correct base sequence before we send them to iGEM. Lukas found that the requirements for Bio LifeSciences was 5-6µL of plasmid DNA at a concentration of 100ng/µL and the primers at a quantity of 10µL and concentration of 3.2pmol/µL. The primers were ordered and the plasmid samples aquilotted in preparation.

The genes of our comparator circuit DNA constructs were finalised within the requirements of GenScript. With three weeks left, we are hoping they will arrive at the end of week 9 or the start of week 10. This will hopefully give us enough time to get into the BioBrick format, send them off to iGEM and characterise and prove their function.

Labs

As the previous transformations of the BioBricks BBa_E0420 (CFP) and BBa_K081014 (RFP) were either unsuccessful or showed contamination so we decided to retry the transformation with a different team member. The procedure used was the one seen on the lab protocol page. In addition, the BBa_K206009 (GFP) BioBrick was transformed. Again, this will also be utilised in characterisation of our BioBricks as these will eventually be ligated to our promoter. The transformants were plated on ampicillin plates of a concentration of 100µg/ml. A negative control of E.coli cells was implemented as well.

Day 2 (21/08/12)

Labs

A message from GenScript confirming the synthesising of the ordered genes!

The transformation was unsuccessful as there was no sign of contamination and no growth showed on any of the plates. A transformation of CFP, RFP and GFP into alpha competent cells was repeated. Some changes were made: positive controls were made (plasmids containing MB within backbones that contain both ampicillin and chloramphenicol resistance). Through some slight confusion, GFP had been plated on ampicillin plates the previous day, as the BioBrick contains only chloramphenicol resistance, it was plated on chloramphenicol plates (25µg/ml) today. The rest of the protocol was unchanged from the previous day. Some of the transformation culture was retained and more LB medium was added and incubated over night.

A growth study involving the PyeaR-GFP (BBa_K381001) BioBrick and alpha cells was planned again but this time involving MB and BM BioBricks as well. We hope to obtain results that will allow us to compare the data sets. The plan is to grow these cells in LB medium without antibiotic resistance in sterile conditions overnight to increase the number of cells. The next day, these will be pelleted down to obtain a larger cell concentration and these samples will be diluted down with LB both till an absorbance of 0.2 is reached. From then on, measurements will be run for 12 hours (Results).

Day 3 (22/08/12)

Labs

Figure 1. B-M/M-B in pSB1C3 plasmid preps, double digested using PstI and SpeI to linearize for later ligation with fluorescent reporter

Following the protocol of the growth study set up the previous day, Khadija, Lukas and Rebecca arrived into the labs bright and early. The full experiment details can be found on the Experiments page.

The transformations of the GFP, RFP and CFP were successful. Growth was observed on all plates except the negative control plates. Some showed greater numbers of colonies than others. The positive control showed lots of viable cells. The GFP plates showed lots of growth, too. The RFP and CFP showed less growth. There were less than 10 colonies on these plates. Inoculations with viable cells from all samples were made into LB media and incubated overnight at 37°C.

A restriction digest was carried out by Khadija to both validate and linearise BM and MB within the iGEM pSB1C3 backbone. It was linearised in preparation of ligating a reporter to the insert (B-M and M-B hybrid promoter). The restriction digest was carried out using PstI and SpeI. The samples were left overnight to fully complete the digestion. From previous experiments which ligated MB and BM into pSB1C3, we used 4 BM's and 4 BMs samples labeled BM1-4 and M-B1-4 respectively. These were used in the digestion (Figure 1.).

The running inoculations of MB and BM BioBricks were miniprepped, following the protocols on the lab protocol page. This produced more plasmid DNA which was nano dropped.

Day 4 (23/08/12)

Labs

The overnight restriction digest was run on an agarose gel (1% w/v) for an hour and a half. The image showed that MB1, MB2, MB4 and BM2 were fully cut and presented with only one band. The smaller excised fragment, which would be only a few base pairs long, was not visible as expected. Some of the samples ran further than exspected and we hypothesised that these might be uncut plasmids (Figure 1.).

BioBrick (BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP) transformants were miniprepped by Lukas to attain pure plasmid DNA. These was stored in the freezer.

The growth study carried out the previous day did not include the lag phase as the starting concentrations of cells were too high. A lag phase only study was carried out. This involved starting measurements at a lower cell concentration (relative to absorbance). The absorbances starting point was chosen at around 0.04, however reaching these values precisely was very difficult due to the error margin of the spectrophotometer being great in this range. Therefore 10µl of cultures grown overnight were pipetted into 1ml of LB broth media in a cuvette. The study lasted for 5 hours with readings taken every hour. Results are shown on the experiments page.

The sequencing primers have arrived! iGEM sequencing primer (VF and VR) stock was made up to a concentration of 100µM by resuspension the stock solution in distilled water. These aquilots were diluted to match the concentration requirements of Bioscience at 3.2pml/µL. Following the arrival and preparation of primers, the plasmid DNA of our BioBricks to be sent for sequencing was prepared. The made DNA was packaged into separate eppendorfs to be sent off. Each eppendorf contained 6µL of the DNA (MB1-4 and BM(1-4) and was labelled accordingly. As there was a delay, these were unable to be sent off immediately and were frozen again until postage.

Day 5 (24/08/12)

Labs

Figure 2. Gel electrophoresis of Isolated plasmids of GFP, CFP, RFP BioBricks, single digested with PstI

Rachel carried out a gel purification (on both MB and BM fragments) using a Promega Wizard kit (Ref: A9281) as described in the lab protocol page. The purified samples were stored in the freezer.

Rebecca transformed BioBrick BBa_K561002 (PFDHF +RFP + TETR) into Bioline Alpha Select Gold Standard cells following the transformation protocol as seen on the lab protocol page. The negative control was simple, untransformed alpha cells. The samples were plated onto agar plates containing chloramphenicol at 25µg/ml.

Khadija nanodropped miniprepped plasmid BioBrick samples of BBa_K206009 (GFP), BBa_E0420 (CFP) and BBa_K081014 (RFP). As several morphologically different colonies had grown an several plates, the samples of these cells were stored in labeled epppendorfs. In total there were 4 different plasmid isolations of CFP, RFP and GFP. These were shown to have high concentrations of DNA ranging from 165ng/µL to 332 ng/µL. Following a nanodrop, two restriction digests were carried out to validate the DNA. One cut at the brick insert at the suffix/prefix, the other digested at a specific restriction site within the backbone. As BBa_K206009 is found in pSB1AK3, there is a HinIII site and BBa_K081014 is in pSB1C3 and contains an EcoRV site. BBa_E0420 contains many restriction sites, but none that can be cut by enzymes that we had in stock. Therefore, a second validation restriction digest was not carried out on this BioBrick. The protocol involved 1µL DNA, 2µL buffer, 0.2µL BSA, 0.5µL of restriction enzyme and 16.3 µL of distilled water. Buffer H was used in the digests with EcoR1, Buffer B for HindIII and Buffer D for EcoRV. These completed digestions were stored in the freezer after 2 and a half hours of digestion ('Figure 2.)