Team:Newcastle/5 August 2010
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{|border=1 | {|border=1 | ||
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- | !'''pSB1C3 | + | ! |
- | + | !'''Biobrick compatible vector pSB1C3''' | |
- | !''' | + | !'''Pspac_oid pormoter''' |
- | + | !'''1st fragment of ''rocF'' CDS''' | |
- | !''' | + | !'''2nd fragment of ''rocF'' CDS)''' |
- | + | !'''3rd fragment of ''rocF'' CDS''' | |
- | + | !'''Double Terminator''' | |
- | + | ||
- | + | ||
- | + | ||
- | !''' | + | |
- | + | ||
- | + | ||
- | + | ||
- | !'''Double | + | |
- | + | ||
|- | |- | ||
|44.0 µl/ml | |44.0 µl/ml |
Revision as of 15:47, 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 | ||
---|---|---|---|---|---|---|---|
44.0 µl/ml | 19.9 µl/ml | 25.0 µl/ml | 30.8 µl/ml | 10.0 µl/ml | 44.2 µl/ml | 9.2 µl/ml | 39.7 µl/ml |
Table 1: Nanodrop spectrophotometer experiment result. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
We found bands in the lane 2, 3, 4, 5 and 6 showing the presence of plasmid in E. coli DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. Also while doing nanodrop experiment, we measured 260/280 nm ratio for all the samples came out to be between 2.0 to 2.4 approximately which shows that there is a high RNA contamination in the samples.
Conclusion
This experiment shows that there is plasmid present in the E. coli DH5α cells but they are present in a very low amount and having high RNA contamination possibly due to the following reasons:
- P1 buffer which contains RNAse might be contaminated.
- RNAse enzyme might have gotten inactive.
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.
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