Team:University College London/LabBook/Week15

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Contents

15.1 Monday 17.09.12

Analytical digest and Gel of the following ligations:


  • BBa_K729004 + BBa_J23119-BBa_0034
  • BBa_K729003 + BBa_J23119-BBa_0034
  • BBa_K118011 + BBA_I746916


Methods

Restriction Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC


Aim - We want to check if the ligations have been successfulccessful


Methods:


Results:


Gel 1 table 1week 15.png


Conclusion: The bands that we obtained on the gel did not correspond to the sizes we were expecting. For most of the samples we could clearly see a band at around 2000 which most probably is the backbone. These confusing results may be due to mistakes during the ligation or the enzyme digestion. Contamination is another possible reason for these results.



Gel 1 table2week15.png

15.2

Tuesday (18.09.12)

Analytical digest and Gel of the following ligations:


  • BBa_K729003 + BBa_J23119-BBa_0034
  • BBa_K118011 + BBA_I712069
  • BBA_I719005 + BBA_I712069
  • BBa_I746909 + BBa_K729007


Methods

Restriction Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC


Gel2table1week15.png


Gel2table2week15.png

Conclusion: We have obtained the expected sizes on for the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation.


Picking Colonies
Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.


Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.


(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)


Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube


Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.

15.3 Tuesday 18.09.12

Aim – We would like to ligate BBa_I746909 to BBa_K729007 to obtain the following construct pCstA+RBS+T7RNAP+pT7+RBS+GFP+TT


Step 1 We minipreped the successful ligation of the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation which is our biobrick BBa_K729007


Step 2 Ligation was performed using the Biobricks BBa_I746909 and BBa_K729007 to generate a Biobrick that contains pcstA-T7RNAP-pT7-GFP-TT.


Step 3 Transformation – we plated 4 plates; plate 1 and 2 were with the transformed BBa_I746909 biobrick and plate 3 and 4 were transformed with our ligation (BBa_I746909+BBa_K729007). We are going to incubate over night at 37C and colonies picking the next day.


Aim 1 – Check the result of transformation. We expect to see growth on plates with transformed biobricks and ligations.


Traformationweek15.png



Results - The table below indicates that there was growth on all of the plates


Plate Samples Volume Inoculated Colony Formation
1 Biobrick BBa_I746909 10ul Yes
2 Biobrick BBa_I746909 90uL Yes
3 Ligation BBa_I746909+BBa_K729007 10ul Yes
4 Ligation BBa_I746909+BBa_K729007 90uL Yes


Conclusion : The transformations have been successful. Colonies will be picked and inoculated into LB media for overnight culture

15-4

Tuesday (18.09.12)



Aim – We would like to ligate BBa_I746909 to BBa_K729007 to obtain the following construct pCstA+RBS+T7RNAP+pT7+RBS+GFP+TT


Step 1 We minipreped the successful ligation of the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation which is our biobrick BBa_K729007


Step 2 Ligation was performed using the Biobricks BBa_I746909 and BBa_K729007 to generate a Biobrick that contains pcstA-T7RNAP-pT7-GFP-TT.


Step 3 Transformation – we plated 4 plates; plate 1 and 2 were with the transformed BBa_I746909 biobrick and plate 3 and 4 were transformed with our ligation (BBa_I746909+BBa_K729007). We are going to incubate over night at 37C and colonies picking the next day.

Wednesday (19.09.12)

Aim 1 – Check the result of transformation. We expect to see growth on plates with transformed biobricks and ligations.


Results - The table below indicates that there was growth on all of the plates


Plate Samples Volume Inoculated Colony Formation
1 Biobrick BBa_I746909 10ul Yes
2 Biobrick BBa_I746909 90uL Yes
3 Ligation BBa_I746909+BBa_K729007 10ul Yes
4 Ligation BBa_I746909+BBa_K729007 90uL Yes


Traformationweek15.png


Conclusion : The transformations have been successful. Colonies will be picked and inoculated into LB media for overnight culture


Step 4 – Inoculating Colonies into a Selective Broth. We had colonies on all the plates. We picked we picked 2 colonies from plates 1 and 2 and 4 colonies from plate 3 and 4. We grow all 12 colonies overnight.The table below indicates the volume of broth and the concentration of antibiotic required.

Picking Colonies
Step 1 – Creating culture media: In a sterile environment, set up X numbers of falcons, each with 5mls of media.


Step 2 - Inoculating Colonies into a Selective Broth:: Add Yul of antibiotic to reach desired antibiotic concentration.


(For Ampicillin this is 50ug/ml, For Kanamycin it is 25ug/ml, for Tetracycline it is 15ug/ml, and for Chloramphenicol it is 25ug/ml)


Step 4 – Selecting a Colony: Select a clear, isolated colony and using an inoculation hoop scoop up a colony onto the tip. Deposit in the falcon tube


Step 5 - Culture: Culture your falcon tubes overnight at a temperature of 37 oC. Leave for no longer than 16 hours.


Falcon tube Samples Broth Antibiotic (50 nu/uL)
1 Biobrick BBa_I746909 Lysogeny Broth Ampicillinn
2 Biobrick BBa_I746909 Lysogeny Broth Ampicillin
3 Biobrick BBa_I746909 Lysogeny Broth Ampicillin
4 Biobrick BBa_I746909 Lysogeny Broth Ampicillin
5 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
6 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
7 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
8 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
9 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
10Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
11 Ligation BBa_I746909+BBa_K729007 Lysogeny Broth Chloramphenicol
12 Ligation BBa_I746909+BBa_K729007 Lysogeny BrothChloramphenicol

Thursday (20.09.12)

Aim - Check if the ligations were successful.

All 12 colonies so we are going to miniprep and nanodrop all of them

Step 1 - Miniprep

Step 2 - Nanodrop

In the table below are summarised all the concentrations obtained from the nanodrop.


Falcon tube Samples Plate Concentration at λ260 (ng/uL)
1 Biobrick BBa_I746909 1 188
2 Biobrick BBa_I746909 1 276.2
3 Biobrick BBa_I746909 2 194.6
4 Biobrick BBa_I746909 2 329.6
5 Ligation BBa_I746909+BBa_K729007 3 233.6
6 Ligation BBa_I746909+BBa_K729007 3 197.7
7 Ligation BBa_I746909+BBa_K729007 3 438.5
8 Ligation BBa_I746909+BBa_K729007 3 337.8
9 Ligation BBa_I746909+BBa_K729007 4 310.3
10Ligation BBa_I746909+BBa_K729007 4 244.3
11 Ligation BBa_I746909+BBa_K729007 4 249.8
12 Ligation BBa_I746909+BBa_K729007 4340.2


stet 3 - We carried out analytical digest of the minipreped DNA

step 4 - Gel electrophoresis of the analytical digest to see if we have the right DNA size


Gel3table1week15.png


L - DNA ladder 10037 bp (5μL)

1 - PstI cut BBa_I746909+PSB1A3

2 - PstI and Xbal cut BBa_I746909+PSB1A3

3 - PstI cut BBa_I746909+PSB1A3

4 - PstI and Xbal cut BBa_I746909+PSB1A3

5 - PstI cut BBa_I746909+PSB1A3

6 - PstI and Xbal cut BBa_I746909+PSB1A3

7 - PstI cut BBa_I746909+PSB1A3

8 - PstI and Xbal cut BBa_I746909+PSB1A3

9 – PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

10 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

11 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

12 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

L DNA ladder 10037 bp (5μL)

13 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

14 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

15 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

16 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

17 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

18 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3


Gel3table2week15.png


L - DNA ladder 10037 bp (5μL)

19 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

20 - PstI cut cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

21 - PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

22 - PstI cut ligation (BBa_K729007+BBa_I746909) + PSB1C3

23 -PstI and Xbal cut ligation (BBa_K729007+BBa_I746909) + PSB1C3


Biobricks Biobrick size (bp) Ligation size (bp) Plasmid backbonePasmid bacbone(bp)Combined size(bp
BBa_K729007+BBa_I746909 2817+906 3731 PSB1C320705801
BBa_I746909 906 PSB1A3 2155 3061


Conclusion - The ligation was successful the bands on the gel correspond to the bands we were expecting.

15-5