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Team:Newcastle/31 August 2010
From 2010.igem.org
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Contents |
Amplification of the plasmid PSB1C3 for rocF BioBrick
Aim
The aim of this experiment is to amplify plasmid linearized pSB1C3 for the construction of rocF BioBrick with the help of a single Phusion PCR using old primers.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol. The details for the single PCR reaction is mentioned below:
PCR
| Tube | Part to be amplified | DNA fragment consisting the part | Forward primer | Reverse Primer | Melting Temperature (Tm in °C) | Size of the fragment (in bp) | Extension time* (in seconds) |
|---|---|---|---|---|---|---|---|
| 1 | Plasmid Vector | pSB1C3 | P1V1 forward | P2V1 reverse | 58 | 2072 approx. | 65 |
Table 1: Table represents a single Phusion PCR reaction where plasmid pSB1C3 is amplified so that it can be ligated together with other rocF fragments with the help of Gibson Cloning method.
- The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
- For learning about the rocF fragments, please refer to the Cloning strategy for rocF.
Discussion
The Phusion PCR reaction was done however, gel electrophoresis and gel extraction will take place today to check whether the fragments have actually amplified or not.
Conclusion
Today, we would be running gel electrophoresis to check the outcome of the PCR reaction and later all the fragments will be ligated with help of Gibson protocol.
Gel extraction of amplified plasmid pSB1C3 for rocF
Aim
The aim of this experiment is to perform gel extraction of the bands containing the amplified fragment of the plasmid vector pSB1C3.
Materials and Protocol
Please refer to: Gel extraction.
Result
For plasmid vector pSB1C3, we used 1.5% agarose gel and run the gel on 75 Volts for better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector.
| Plasmid Vector pSB1C3 | |
|---|---|
| Size of the Fragment (in bp) | 2072 approx. |
Table 2: Table represents the size of the plasmid vector pSB1C3 represented as bands on the gel.
Discussion
We found bands of appropriate size and cut it out and placed the part of the gel in a 2ml eppendorf tube.
Conclusion
Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
Amplification of the parts for Subtilin Immunity BioBrick
Aim
The aim of this experiment is to amplify the four parts for the Subtilin Immunity BioBrick using Phusion PCR again but using the original primers that were ordered and hydrated. (Before this could be done, four of the primers, 1-T1_for, 2-T1_rev, 1-P1_for and 2-P2_rev, had to be re-hydrated.)
For more information about the subtilin immunity BioBrick, please see the cloning strategy for subtilin immunity (Link)
Materials and Protocol
Please refer to Phusion PCR for Phusion PCR protocol. (Please refer to DNA Re-hydration for DNA Re-hydration protocol.)
The details for the four PCR reactions are included in the table below:
| Tube | Part to be amplified | DNA fragment consisting the part | Forward primer | Reverse Primer | Melting Temperature (Tm in °C) | Size of the fragment (in bp) | Extension time* (in seconds) |
|---|---|---|---|---|---|---|---|
| 1 | Plasmid Vector | linear PSB1C3 (cut with HindIII) | P1V1 forward | P2V1 reverse | 53.3 (53) | 2046 + | 70 |
| 2 | Promoter and RBS (pVeg-SpoVG) | BioBrick Bba_K143053 | P1P1 forward | P2P2 reverse | 51.7 (51) | 139 + | 15 |
| 3 | spaIFEG Gene Cluster | B. subtilis ATCC 6633 | P1S1 forward | P2S1 reverse | 53.0 (53) | 2753 + | 110 |
| 4 | Double terminator | pSB1AK3 consisting BBa_B0014 | P1T1 forward | P2T1 reverse | 50.9 (51) | 116 + | 15 |
Table 2: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts will be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
- The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
Results, Discussion and Conclusion
Gel electrophoresis and Gel extraction will be carried out if the bands are successful.
yneA
Single Digest
Aim
To do single digest of pGFPrrnB with Pst1 and Nhe1 to test if the enzymes are contaminated.
Materials and Protocol
Please refer to restriction digest and gel electrophoresis.
Results
Setting up Overnight Cultures
Aims
To prepare overnight cultures for minipreps of yneA, pGFPrrnB and pSB1C3, and also cultures for B. subtilis transformation.
Materials and Protocol
Please refer to growing an overnight culture.
   
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