Plasmid Miniprep Experiment

Aim

The aim of this experiment is to extract plasmid DNA pSB1C3, pSB1AK3 and plasmid containing lacI Biobrick from E. coli DH5α cells with the help of Qiagen miniprep kit and confirming the extraction with the help of nanodrop experiment.

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

Newcastle_020810_gel.png

• 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)

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, and 5 showing the presence of plasmid in E. coli DH5α cells. From tht 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

This experiment shows that there is plasmid present in the E. coli DH5α cells but they are present in a very low amount possibly due to plasmid shuffle which could have occurred during overnight growth in the cultures which did not contain antibiotics against which plasmid provides resistance to the cell.