LIGATION+Troubleshooting

=This week (October 3-7), we attempted to do ligation. In hand, we already had alot of ligase buffer from last year. Last month, we ordered linearized vector, ligase enzyme, and bacterial cells from Genotyp company.=


 * 1) = Using a PCR made with Invitrogen Taq polymerase, we performed the first ligation. We used about ~6uL of remaining PCR product, 4uL of vector and 1uL of undiluted ligase enzyme, all in ligase buffer (Annemarie). We let the reaction sit for 30 min before performing bacterial transformation. We also did a transformation with a positive control using DNA template (plasmid) instead of a ligation product (Paige). All transformations were done with the bacterial cells recently sent by Genotyp. Transformations were allowed to grow overnight in antibiotic-free SOCS medium, and were plated on ampicillin-arabinose agar plates the next day by Aprell. Here are the results from the plate growth: =
 * == __The ligation with the PCR product produced no colonies__.This could be due to inactivity of the ligase enzyme, the short length of the ligation time (i.e., 30 min), or a very low concentration of the linearized vector (i.e., 6-7 ng/uL). ==
 * == __The transformation with the positive control produced several hundred GFP-positive colonies__. This means that the bacterial cells were competent, and were not the cause of the ligation-transformation result. Also, the new agar plates were not a problem (i.e., arabinose or ampicillin). ==