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Team:Newcastle/3 August 2010
From 2010.igem.org
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Contents |
Plasmid Miniprep Experiment
Aim
The aim of this experiment is to extract plasmid DNA pSB1C3 and pSB1AK3 from E. coli DH5α cells using the Qiagen miniprep kit and analysing with 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. NOTE: 10 µl of RNAse A have been added into the current P1 buffer from Qiagen.
Result
Figure 1:Gel electrophoresis of the PCR products
- 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 plasmid containing lacI (No. 1)
- Lane 6: Extraction of pSB1AK3 plasmid containing double terminator (No. 1)
- Lane 7: 1kb DNA ladder
| pSB1C3
(No. 1) | pSB1C3
(No. 2) | pSB1C3
(No. 3) | lacI
(No. 1) | Double terminator
(No. 1) |
|---|---|---|---|---|
| 92.1 µl/ml | 110.0 µl/ml | 110.5 µl/ml | 246.1 µl/ml | 246.7 µl/ml |
Table 1: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
Bands around 3000 bp were observed in lanes 2, 3 and 4, thereby indicating the correct products. Lane 5 contain the LacI plasmid,therefore it should be bigger in size as compared to the double terminator plasmid which is in lane 6. Therefore lane 5 might not contain the LacI plasmid. From the nanodrop results, the ratio of 260.280 nm ranged from 1.7 to 1.85 and the concentration ranged from 92.1 µl/ml to 246.7 µl/ml. These results indicated that the DNA extracted have low RNA contamination and the samples are pure.
Conclusion
Extraction of pSB1C3 plasmid and pSB1AK3 plasmid containing the double terminator have been successful. As well as the addition of RNAse into the P1 buffer solved the problem of RNA contamination.
   
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