So a modified version of my protocol worked:
100uL CaCl2 100mM
1uL 1ng GFP Biobrick practice plasmid
1 colony of 16hr HB101
10min 4C
30sec 42C
2min 4C
30min 37C
Plated on LB Amp IPTG overnight.
I got colonies (about 30) but during a time trial experiment with 4 repeats of 15-45sec at 42C I got mixed results. I believe this is due to the variation in colony pickup and dispersal in the transformation solution. I'm going to do some backlogged leg work and find the CFU/mL of my strain and grow conditions and only use standardized liquid suspensions. Using a pipette tip to pick and place colonies is just too variable. Do you guys think the outgrowth at 37c for 30min would actually be detrimental. Dr. Knight stated earlier that this may be an issue especially since this new protocol does not add any SOC or LB Broth during outgrowth. I mean the bacteria would need some time to express the AmpR before their next mitotic event....but again that's just theory. Someone said they plate directly after cold recovery and skip outgrowth. I guess by the time some cells, which started their cycle upon plating, multiply the AmpR would already be in effect?
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
100uL CaCl2 100mM
1uL 1ng GFP Biobrick practice plasmid
1 colony of 16hr HB101
10min 4C
30sec 42C
2min 4C
30min 37C
Plated on LB Amp IPTG overnight.
I got colonies (about 30) but during a time trial experiment with 4 repeats of 15-45sec at 42C I got mixed results. I believe this is due to the variation in colony pickup and dispersal in the transformation solution. I'm going to do some backlogged leg work and find the CFU/mL of my strain and grow conditions and only use standardized liquid suspensions. Using a pipette tip to pick and place colonies is just too variable. Do you guys think the outgrowth at 37c for 30min would actually be detrimental. Dr. Knight stated earlier that this may be an issue especially since this new protocol does not add any SOC or LB Broth during outgrowth. I mean the bacteria would need some time to express the AmpR before their next mitotic event....but again that's just theory. Someone said they plate directly after cold recovery and skip outgrowth. I guess by the time some cells, which started their cycle upon plating, multiply the AmpR would already be in effect?
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Sebastian Cocioba
Sent: 8/20/2013 1:37 PM
To: Koeng
Subject: RE: Fwd: [DIYbio] Thermal Cycler Based Bacterial Trasformation