Restriction of our vector pJET1.2 in order to ligate each of our genes to the vector (done for each gene separately)
Restriction was done with the same restriction enzymes for both the genes and vector (BamHI and Xhol)
Ligation and transformation to DH5α E.coli competent cells
these cells multiply said plasmid for the purpose of not having to start from the beginning in case a mistake is made during later steps
August-September
Restriction of our genes that were ligated to the vector in order to ligate them to a new vector, which was pBlueScript II SK (+) using the same restriction enzymes as above
Transfer was done through restriction of the plasmids that had the pJET1.2 vector and our genes and through Gel Electrophoresis, the desired fragments were cut and purified
in this case, our desired fragments were those of our genes
Ligation and transformation of the genes and pBluescript to DH5α E.coli competent cells
After each transformation, the cells were platted out in LB-medium Ampicillin Agar plates. The developing colonies were then picked to produce cultures.
from those cultures we obtained our plasmids using a kit provided by Promega
After each step, we sent our plasmid products for sequencing in order to ensure that we were on the right path and everything was it should be
Another method used to check our progress was check-PCR, where through amplification and then Gel Electrophoresis, we could determine if our product had the correct size according to which step we were at that point in time
Mistakes made during lab work:
Communication problems between the lab members, lead to mistakes during lab work
for example, due to miscommunication, the wrong restriction enzymes were used
many different people were coming to the lab who had different amounts of information about what was happening at the lab
this led to there being a lot of probes that were not clear on what they contained
for example, a probe that was used did not have the vector as previously thought
also led to using an insert that was known to have worked for later steps
The lab book was also not thoroughly written, leading lab members to not understand what was previously done.