IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-19: Difference between revisions
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===Site Specific Mutagenesis Attempt 4=== | ===Site Specific Mutagenesis Attempt 4=== | ||
Dave and Peng are going to beat this thing until it works. | Dave and Peng are going to beat this thing until it works. | ||
'''Vials''' | |||
8-5, 3-9, 6-4, 10-7, -t for each (dave), -v for each (peng), cat gene as a positive control, -t | |||
For cat gene, thrbc_cat_up, thrbc_cat_lo | |||
'''New protocol, based on the Silver protocol on OpenWetWare:''' | '''New protocol, based on the Silver protocol on OpenWetWare:''' | ||
Revision as of 08:45, 19 July 2006
Site Specific Mutagenesis Attempt 4
Dave and Peng are going to beat this thing until it works.
Vials
8-5, 3-9, 6-4, 10-7, -t for each (dave), -v for each (peng), cat gene as a positive control, -t
For cat gene, thrbc_cat_up, thrbc_cat_lo
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, 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 8-5, cat gene, negative
#*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
