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Contents 1 Plasmid Miniprep Experiment 1.1 Aims 1.2 Materials and Protocol 1.3 Result 1.4 Discussion 1.5 Conclusion 1.6 Solution for the problem

Plasmid Miniprep Experiment

Aims

The aim of this experiment is to extract plasmid DNA from pSB1C3 and pSB1AK3 from E. coli DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.

Materials and Protocol

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

Result

pSB1C3 (No. 1)	pSB1C3 (No. 2)	pSB1C3 (No. 3)	pSB1C3 (No. 4)	lacI (No. 1)	lacI (No. 2)	Double terminator (No. 1)	Double terminator (No. 2)
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 results. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

In our experiment, the concentration of our plasmid DNA range from 19 µg/ml to 44 µg/ml. This value is lower than the standard value of 150 µg/ml for plasmid DNA extraction. The 260/280 nm ratio for all the samples is in the range of 2.0 to 2.4. These indicated that our sample is contaminated with RNA and the purity of our samples are low.

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.