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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 [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] 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 [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] 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 contamination and the samples are pure.
Conclusion
Extraction of pSB1C3 plasmid and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] plasmid containing the double terminator have been successful. As well as the addition of RNAse into the P1 buffer solved the problem of contamination.
   
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