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: Minipreps for Qiagen miniprep protocol, Nanodrop Spectrophotometer for nanodrop protocol and Restriction digests for restriction digestion protocol.

Result

• Lane 1: 1kb DNA ladder
• Lane 2: Extraction of pSB1C3 plasmid (No. 1)
• Lane 3: Extraction of pSB1C3 plasmid (No. 2)
• Lane 4: Extraction of pSB1C3 plasmid (No. 3)
• Lane 5: Extraction of pSB1C3 plasmid (No. 4)
• Lane 6: Extraction of plasmid containing lacI (No. 1)
• Lane 7: Extraction of plasmid containing lacI (No. 2)
• Lane 8: Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
• Lane 9: Extraction of pSB1AK3 plasmid containing double terminator (No. 2)

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:

1. P1 buffer which contains RNAse might be contaminated.
2. RNAse enzyme might have gotten inactive.

Solution for the problem

1. 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.
2. If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.