Inclusion of restriction sites ...ERRATUM
andrei.popov at bbsrc.ac.uk
Sun Nov 3 21:15:21 EST 1996
Ashok Aiyar wrote:
> There are several possible explanations. The one I favor is
> that Klenow left ends that were considerably more ragged than
> predicted .... which certainly explains the complete lack of
> clones in my cloning. At the time we used cells made competent
> by treatment with CaCl2, and my recollection is that we obtained
> transformation efficiencies in the range of 1x10E6 colonies per
> microgram of DNA.
> If you are able to clone with this approach, my suspicion is
> that either you have used a significantly different ratio of
> Klenow units/ends of DNA, or that your transformation efficiency
> allows you to detect an extremely rare ligation event.
Well, to be precise, that's what I do:
1. PCR products (600 bp 5'RACE) are purified on Glass Milk (GENECLEAN II
BIO 101) elute in 10 mkl of water; the amount was roughly 400 ng
2. to create EcoRI ends: 0.1 mM dCTP/dGTP end concentration,
1/10 volume 10x Klenow buffer (0.5 M Tris-Cl; 0.1M MaCl2),
0.5 mkl 5 U/mkl Klenow fragment; incubate 10 min at RT.
3. Extract with phenol once, spin, load on 1% agarose gel
4. pUC19/EcoRI load on the same 1% gel
5. Cut the bands from the gel, purify on GENECLEAN II.
6. Estimate the amount of DNA eluted from the glass.
7. Ligation: in 10 mkl assemble 10 ng pUC19/EcoRI
10 (or 30) ng of the PCR fragments/RI
0.2 mkl NEB T4 ligase (5 U/mkl)
8. leave on the bench overnight
9. heat the ligation mix for 10 min at 70C
10.Transform into Inoue competent cells (they were prepared around 9
months ago and stored at -70C; the original efficiency of
transformation was in the range of 1x10E8 per mgk of uncut pUC- cant
remember now precisely)
no ligase- 50 colonies
+ligase no insert thousands of colonies, almost all blue
+ ligase + insert thousands of colonies, around 3-5% are white
12.Preps were made from 30 white colonies: in 21 of them the 600 bp
insert could be cut out with EcoRI. In several more preps the linear
vector was bigger (probably the insert went in with killing one EcoRI
site- I did not check them).
13. sequencing confirmed different V lambda genes (5'RACE products).
right now I am tired of typing
> P.S. The PCR product I was attempting to clone, did clone when
> self-ligated (after 5' phosphorylation) followed by digestion
> with EcoRI.
That must have been a REAL miracle!
If you have:
aattcNNNNNNNNNgaatt + aattcNNNNNNNNNgaatt
ttaagnnnnnnnnncttaa + ttaagnnnnnnnnncttaa
plus phosphatase plus T4 ligase and you got a digestable EcoRI site?!
star activity or what?
With best regards,
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