IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-25

Miniprep of reinoculated Hetmann Six

Eluted in 50 uL of H2O. Nanodrop readings:

HH1: 111.5 ng/uL HH2: 47.5 ng/uL HH3: 19.7 ng/uL HH4: 64.0 ng/uL HH5: 54.2 ng/uL HH6: 44.3 ng/uL

Site Specific Mutagenesis Attempt 5

Peng f-ed up the primers...

Vials , 3-10, 9-8, 6-7, 5-4, -t for each, cat gene as a positive control.

For cat gene, pbc_ks+, kt_b, kt_bb primers - expected size ~1.1kb

New protocol, based on the Silver protocol on OpenWetWare:

 5 µL 10x ThermoPol buffer (Vent)
 1.0 µL 10 mM dNTPs 
 2.5 µL 20 µM forward primer 
 2.5 µL 20 µM reverse primer 
 1 µL plasmid DNA
 1 µL Vent DNA polymerase 
 37 uL dH20

PCR Time for 3-9, 6-4, 10-7, cat gene (peng), negative for each

#*95 °C for 2 min. (melt) 
#*95 °C for 0.5 min (melt) 
#*57 °C for 0.5 min. (anneal) 
#*74 °C for 1.5 min. (extension)
#* Go to step 2 (30x)
#*74 °C for 5 min. (final extension; modified from Silver protocol)
#* Keep at 4 °C forever

Total time:  82 minutes +/- some = 90 min

PCR Time for 6-7, cat gene (dave), negative for each

#*95 °C for 2 min. (melt) 
#*95 °C for 0.5 min (melt) 
#*57 °C for 0.5 min. (anneal) 
#*74 °C for 2.5 min. (extension)
#* Go to step 2 (30x)
#*74 °C for 5 min. (final extension; modified from Silver protocol)
#* Keep at 4 °C forever

Total time: 111 minutes +/- some = 120min

Results Expected: 3-10: 209bp
9-8: 370bp
6-7: 2402bp
5-4: 80bp