Team:Newcastle/5 August 2010
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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: | 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. | # Primer sequences could be incorrect. | ||
+ | # Melting temperature could be incorrect. | ||
# Plasmid pMutin4 could have degenerated due to long term storage. | # Plasmid pMutin4 could have degenerated due to long term storage. | ||
Revision as of 15:58, 5 August 2010
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Contents |
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.
- Melting temperature 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
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