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

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 for a better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector.

Table 2: Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.

Discussion

We found bands of appropriate sizes in their respective lanes.

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:

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.