Invitrogen TOPO TA Cloning - 505bp insert.
1. Using the vector for LaZ/blue white screening - I'm getting many blue colonies with a few white colonies and a majority do not contain my insert.
2. Using the vector with the lethal gene, I'm getting a few large white colonies (positive clones) with many small white colonies (false positives and they are not satellite colonies).
Things I've considered and ruled out as a problem:
1. Insert lacking A overhangs - I've used fresh PCR product, gel purified product and I've tried 10,15 and 20min for my final extension along with using a 1.5min extension during cycling.
2. Molar Ratio: I've tried vector:insert ratios of 1:1, 1:3, 1:5. Along with using just 0.5, 1 or 2ul of fresh PCR product (gel shows single band).
3. LB Kan plate: antibiotic is good
I ran the control with the kit - generate control PCR product (750bp), ligate and transform = many blue colonies and few white positive clones. => low efficiency.
LAST TROUBLESHOOTING IDEA - POSSIBLE SELF LIGATION OF VECTOR
1. Ligation Step, is it critical to add components of ligation mixture in a certain order and kept at a certain temperature? Add salt and insert then vector? Adding salt and vector first at RT may allow for self ligation before insert is added?
2. Should this mix be keep on ice while preparing until everything is added then place at RT for incubation?
3. How much to mix ligation? Very small volume and hard to tap side of tube, pipette up and down to mix?
EpigeneticistMember Since 06 Nov 2012
Offline Last Active May 17 2013 04:47 PM
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Epigenetics, Genetics, Cell Signaling, Cancer Biology