I used T4 DNA ligase to ligate an insert has size about 2000 base pair into a vector has size 12.7 Kilo bases. I do everything possible to get colonies before I have contacted Biolab inc. and they recommended 10-Beta compretent cell. So, again i will be pleased if someone has this strain.
Gonzalo Bidart
If you couldt find DH10B E.coli, you can use this protocol for make ultracompetent E.coli with any standard strain.
1) Inoculate 25 ml SOB medium DO(initial)= 0,05 and grow at 19ºC to DO(final)=0,5 (usually between 24-36hs)
2) Centrifugue 4000 rpm 10min a 4ºC.
3) Resuspend in 8ml of cold TB. Leave 10min on ice.
4) Centrifugue 4000 rpm 10min a 4ºC.
5) Resuspend softly in 2ml of cold TB and 140μl DMSO (keep at -20ºC over night before use it).
Good luck !!
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