Gel Electrophoresis for Amplified Pspac_oid promoter and lacI

Aim

The aim of the experiment is to perform gel electrophoresis for the two PCR reactions viz. lacI and Pspac_oid promoter which took place yesterday 5th August, 2010 and thus confirm that they were successful.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

• Lane 1: 1kb DNA ladder
• Lane 2: Plamid pMutin4 containing lacI
• Lane 3: Plamid pMutin4 containing Pspac_oid promoter
• Lane 4: 100bp DNA ladder

Table 1: Table represents the size of the fragments represented as bands on the gel in their corresponding lanes.

Discussion

We found a band in the lanes 2 of the correct size but lane 3 did not contain any band.

Conclusion

This experiment shows that the PCR reaction was successful for all the fragments apart from Pspac_oid promoter which was present in the lane 3 and did not show any band. This could be because of the following problems:

1. Primer sequences could be incorrect.
2. Melting temperature could be incorrect.
3. Plasmid pMutin4 could have degenerated due to long term storage.

lacI ligation (repeat)

Aim

We aim to repeat the ligation from week 12-17th July.

Materials and Protocols

Please refer to ligation for protocol. However, we did not do a positive control for this experiment.

Discussion

On Monday we will transform E.coli with the ligation product.

Results

Please refer to 09.08.10 for results.

lacI and pVeg spoVG Transformation

Aim

We aim to transform competent E.coli DH5α with pVeg spoVG and lacI in preparation for Gibson Cloning.

Materials and Protocol

Please refer to transformation for protocol.

Discussion

We expect cells transformed with lacI and pVeg to survive on the ampicillin plates.

Conclusion

Please refer to 09.08.10 for results.