pET15b ligation probs.

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Fri Aug 4 09:38:15 EST 1995


 In article <3vqqrr$f1b at lastactionhero.rs.itd.umich.edu> 
 jpsmith at umich.edu (James Smith) writes:

| I have posted here before and have corresponded with some of you via
| email, but I'm still having ligation problems.  I have recently switched
| restriction sites in hopes that I may have better luck.  I have been 
| successfully amplifying a 1.5kb DNA fragment via RT-PCR with a Bam HI 
| restriction site included in the primers, 3 bases from the end of the
| fragment (according to NEB's catalog, Bam HI appears to be a good end-cutter).
| I've been digesting the plasmid with Bam for 3h (checked on gel for
| complete linearization) and then treating with CIAP.  I've been using
| Bio-Rad's Prep-a-Gene kit to purify the plasmid.  The DNA insert was
| digested for 3 h with Bam and also purified with Prep-a-Gene. 
| 
| My ligation mix has included DNA & plasmid in a 3:1 (approx.) ratio.  I've
| been using 2 U of T4 DNA ligase (BM) and the included 10x ligase buffer.
| My ligase appears to be good as it ligated a HindIII lambda digest ladder
| in an hour at RT.
| 
| Following an hour ligation, I ran 10 uL of the 30 uL ligation mix on a gel
| and noted a fairly intense smear from my 1.5kb fragment up to a point 
| corresponding approximately to the location of circular plasmid with no
| insert (although no *distinct* bands were observed).  The smear continued
| up beyond the location of linear plasmid, but was less intense in this 
| region.  I attempted transformation with this ligation rxn, but no colonies
| were observed (transformation was successful on the pUC18 control plates).
.
<rest deleted>

Jim:

I'd like to suggest a few things that might help your ligation with single
inserts. When using a binding matrix like Bio-Rad Prep-A-Gene, I like to
put BOTH linearized plasmid AND insert DNA together BEFORE binding. Then,
when eluting, make sure the pellet is dry (I use roto-vac) and add about
12 ul of H2O -> heat to 55-60 C 5 min. in a water bath, flick the tube with
your finger nail REAL hard, and put it back for another 5 min. at the same temp.
Flick again, spin, and put an ice to anneal the ends. Then, remove 8 ul for
your ligation, + 1 ul 10x ligation buffer + 1 ul ligase. Incubate overnight at
16 C, and then transform using the entire mix. BTW, this method is on the FAQ
list. It hardly ever fails me. Also, if you are using tetracycline as your
marker, express a long time.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
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