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Expression And Characterization Of Grb14 During Zebrafish Embryogenesis

2187 words - 9 pages

5.1 Colony PCR
Colony PCR was used to screen for plasmids containing a desired insert directly from E. coli colonies. Primers are used which generate a PCR product of known size. Hence, any colonies which give rise to an amplification product of the expected size are likely to contain the correct DNA sequence.
The main purpose of conducting colony PCR is to make sure it indicated the presence of the insert and predicting its size. Besides, the plasmid used in this experiment PGEM- T Easy Vector is cut with EcoRV and added with 3’ terminal thymidine at both ends. The T-overhang prevented the recircularization of the vector. Even so, some of the plasmid will self-ligate (blunt-ended ligation) during ligation. This happens when T-overhang have degraded due to the introduction of nucleases. Thus, plasmid without insert will also be transformed into the bacteria. Therefore, colony PCR can be used to screen and shown no result when any of the above conditions is not met. A good transformant that carrying the plasmid with insert will show band with expected size.
After running colony PCR as shown in figure 3, the product size obtained from agarose gel electrophoresis (AGE) was between 750-500 bp which corresponded to the expected amplicon size, which was 725 bp (Lee, 2008). The LB broth culture which corresponded to the correct result of colony PCR was further prosecuted for plasmid minipreparation. Alternatively, LB broth culture with positive clone can be added with 50% glycerol and stored in -80 °C freezer.

5.3 Plasmid Miniprep System (PureYieldTM)
Plasmid DNA was purified from overnight cultures of E. coli with the PureYieldTM Plasmid Miniprep System. The PureYieldTM Plasmid Miniprep System isolated high-quality plasmid DNA for use in eukaryotic transfection and in vitro expression experiments. Silica membrane column present in the system provided a more efficient method for plasmid DNA purification. Plasmid DNA can be purified in less than 10 minutes, depending on the number of samples processed, greatly reducing the time spent compared to silica resin or other membrane column methods.
The plasmid DNA that has been extracted using a Plasmid Minipreparation kit was believed to be very efficient since it a robust and quick method for isolating Plasmid DNA. The plasmid DNA that has been purified was proceed by loading 5 microliter of the plasmid DNA into the gel to make sure it is properly extracted. In figure 4, the plasmid was at position between 2000 bp and 2500 bp ladder to determine it size. However, the plasmid DNA expected size should be 3015 bp plus 725 bp of insert making it 3.7 kb in size. The main reason for the plasmid DNA to sit in this size is due to their owned shape. Plasmid DNA contained three different shaped that is supercoiled, nicked and linear. Supercoiled plasmid DNA moves slower compared to linearized plasmid in AGE. The only way to determine the size of plasmid is using restriction enzyme that cuts the plasmid in on...

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