Gel Electrophoresis for the Amplified Fragments of rocF

Aim

The aim of the experiment is to perform gel electrophoresis for all the 6 PCR reactions which took place yesterday 4th August, 2010 and thus confirm that all 6 PCR reactions were successful.

Materials and Protocol

Please refer to: Gel electrophoresis.

Result

Lane 1: 1kb DNA ladder
Lane 2: BioBrick compatible vector pSB1C3
Lane 3: Pspac_oid promoter
Lane 4: 1st fragment of rocF CDS
Lane 5: 2nd fragment of rocF CDS
Lane 6: 3rd fragment of rocF CDS
Lane 7: Double Terminator
Lane 8: 1kb DNA ladder

	Biobrick compatible vector pSB1C3	Pspac_oid pormoter	*1st fragment of rocF* CDS**	*2nd fragment of rocF* CDS)**	*3rd fragment of rocF* CDS**	Double Terminator
Size of the Fragment (in bp)	2072 approx.	106 approx.	246 approx.	597 approx.	125 approx.	116 approx.

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

Discussion

We found bands in the lanes 2,4,5,6 and 7 of the correct sizes 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:

Primer sequences could be incorrect.
Plasmid pMutin4 could have degenerated due to long term storage.

Solution for the problem

If P1 buffer of the Qiagen miniprep kit is contaminated, then use a different kit. We have Promega miniprep kit which will be used tomorrow.
If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.

Concrete Tensile Splitting Test

Team:Newcastle/5 August 2010

From 2010.igem.org

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