efficient blunt ligation?
hdang at channel.neusc.bcm.tmc.edu
Tue Jul 4 17:17:32 EST 1995
>hdang at channel.neusc.bcm.tmc.edu (hong dang):
>> Just did this with EcoRV digested vector and PCR fragment: gel purify
>> fragment with QIAquick kit, SAP (shrimp alkaline phosphatase, USB) 1 unit
>> for 40 min at 37C then heat inactivate at 65C for 15-20 mins; ligate at
>> 16C overnight with 3:1 of I:V, 400 units T4 ligase, left on the bench
>> (r.t.) in the morning, transform in the afternoon; next day: vector alone
>> 9 colonies, with insert, about 1000.
>You were a very lucky man Hong, but do you think you should tell
>such lies on the internet.
Maybe I should clarify a bit:
1) I did start with 2-3 ug of plasmid (about 5.6 kb) and 2-3 molar fold excess
of EcoRV fragment (300 bp) from 700 bp PCR product obtained using Pfu
polymerase. So this may not qualify as "EFFICIENT" blunt ligation.
2) T4 DNA ligase was concentrated version from NEB. All reactions were done in
a total volume of 20-40 microliters.
3) Final transformation rxn should contain about 200 ng DNA per tube (did
not check but I used half of the products obtained from the previous step
in each stage, e.g. 10 ul of 20 ul ligation mix to transform).
4) I don't work for any of the companies mentioned in any of my posts, and I am
responsible for all contents of my messages.
Still wondering why it is so outrageous that this could actually work?
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