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Team:Newcastle/2 August 2010
From 2010.igem.org
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Plasmid Miniprep Experiment
Aims
The aim of this experiment is to extract 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 expected value of approximately 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. This indicates that our sample are of low purity.
Conclusion
We are able to obtain plasmid DNA, however the concentartion and the purity of the samples are low. This could be due to the following reasons:
- The buffers might have been contaminated with RNA.
- RNAse enzyme might have degraded over time.
Solution for the problem
- If the buffers of the Qiagen miniprep kit is contaminated, then we will be using the Promega miniprep kit.
- If the RNAse enzyme was degraded, then we will be adding 10 µl of fresh RNAse into the P1 buffer.
   
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