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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 Qiagene.
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 experiment result. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
We found bands in the lane 2, 3, 4 having the correct size, which is around 3000 bp. 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 experiment we found that the plasmid DNA extracted is present in high quantity and is of good quality as the ratio of 260/280 nm ranged from 1.7 to 1.85 for the above mentioned plasmid DNAs.
Conclusion
Extraction of pSB1C3 plasmid and pSB1AK3 plasmid containing double terminator have been successful.
   
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