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

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.