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

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.