Most of the protocols used in this research project were obtained from the molecular biology lab manual written by Campbell (1988). Further information for all these standard protocols can be found at the appendix section. Protocols that were modified or changed to accomodate the specificity of my experiments are mentioned below.
A. Polymerase Chain Reaction (PCR)
Primers were constructed to flank the sequence to be amplified. There are 20 nucleotides in the forward primer (CAA CAC TCC GGC TGC CGT TG) while the reverse primer has 30 nucleotides (ATT GGG TAC CCA TGC CAT TGC TTT ACA GCG). The 30-nucleotide reverse primer contains the polyadenylation signal and a Kpn I restriction enzyme cut site. Experiments showed that PCR using 5 ml of 10X buffer with 15 mM Mg2+ and 2 ml of 25 mM MgCl2 provided optimal Mg2+ concentrations (Figure 8). All PCRsí were performed on the PCR thermal cycler using the the GA1 program (95° C for 5 minutes, 95° C for 1 minute, 55.0° C for 1 minute, 72° C for 2 minutes, previous step repeated for 29 cycles, maintained at 20° C).
B. Qiagen plasmid-preps
Qiagen maxi and midi preps were performed on construct A and pMN56 respectively. For the maxi-prep, 100 ml of overnight cell culture was grown, maxi-prepped and the pellet was dissolved in 150 ml of TE (Tris-EDTA) buffer. For the midi-prep, 25 ml of overnight cell culture was grown, midi-prepped and the pellet was dissolved in 100 ml of TE buffer. Concentrations of the mini-preps were determined via a spectrophotometer set at a wavelength of 260 nm. A Beckman J2-21 centrifuge (JA-20 rotor) was used in all the steps that required centrifugation.
C. Quick screen gel electrophoresis
The quick screen gel electrophoresis was used to identify transformed colonies that contained the desired plasmids without having to carry out individual plasmid mini-preps. Cells were grown in the incubator for approximately 2 hours before being lysed by the 2X Quick Dye that contained 20% SDS. These lysed cells were loaded on to agarose gels and gel electrophoresis was carried out to determine the presence of the desired plasmid.
D. Autolysin preparation
Autolysin is needed for Chlamydomonas to shed their cell walls prior to fusion of the gametes. Autolysin was prepared by growing R3+ and NO- Chlamydomonas strains on TAP (Tris-Alkaline-Phosphate) plates. These two strains were then flooded with NFHSM, mixed together and allowed to mate for 20 minutes. A Beckman J2-21 centrifuge (JA-20 rotor) was used to pellet the cells at 9500 rpm and the supernatant which contained the autolysin was collected and stored in a -70° C freezer.
E. Transformation of construct A in pmh1 cells
Chlamydomonas cell cultures were grown in TAP medium until they reached a density of roughly 1.5 x 106 to 3 x 106 cells/ml. An aliquot (2 ml) of autolysin was then added to the cell culture to facilitate shedding of cell walls. Cell wall loss was tested with a 0.2% NP-40 detergent. For co-transformations of pmh1 cells, a nit1 selectable marker was. All transformations used 300 mg of size 6 (425-600 microns) glass beads obtained from Sigma (G9268). Centrifugation of cells were performed by a Labofuge 400 clinical centrifuge at 1435 rpm (700 g).