"
Team:University College London/LabBook/Week15
From 2012.igem.org
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
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:
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.
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
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
Conclusion: We have obtained the expected sizes on for the pcstA (BBa_K118011) +RBS-T7RNAP (BBa_I712069) ligation.
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.
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 |
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.
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 |
| 10 | Ligation BBa_I746909+BBa_K729007 | Lysogeny Broth | Chloramphenicol |
| 11 | Ligation BBa_I746909+BBa_K729007 | Lysogeny Broth | Chloramphenicol |
| 12 | Ligation BBa_I746909+BBa_K729007 | Lysogeny Broth | Chloramphenicol |
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 |
| 10 | Ligation BBa_I746909+BBa_K729007 | 4 | 244.3 |
| 11 | Ligation BBa_I746909+BBa_K729007 | 4 | 249.8 |
| 12 | Ligation BBa_I746909+BBa_K729007 | 4 | 340.2 |
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
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
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 backbone | Pasmid bacbone(bp) | Combined size(bp |
|---|---|---|---|---|---|
| BBa_K729007+BBa_I746909 | 2817+906 | 3731 | PSB1C3 | 2070 | 5801 |
| BBa_I746909 | 906 | PSB1A3 | 2155 | 3061 |
Conclusion - The ligation was successful the bands on the gel correspond to the bands we were expecting.
