IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

CIP is the problem?

R. Jayakumar jakku at mrna.tn.nic.in
Tue Jan 11 03:46:51 EST 2000


hi
    I too faced similar problems in making partial genomic libraries, when I
used CIAP.  Nowadays I don;t use CIP and there is no problem.  I get good
transformed clones.  Maybe you should try checking whether your gel elution
is the problem. Mostly, I find that traditional techniques like freezing, or
elution works out better than many kits do.  Anway I use the magic resin kit
by GIBCO.
    You should also try checking the DNA on an agarose gel after each step;
ie. afte elution, and after ethanol precipitation.  contrary to claims, I
still find ethanol precipitation at -70C for 30' to be the best rather than
at Room temp.
    You should try to exactly estimate the vector and insert concs. by
visual examination rather than by spectrophotometer observations.  Specs
always gives a very erroneous reading and they are not consistent.  it is
very important that you don't do the following while ligationa nd
transformation
    1. Always use only vector:insert conc as 3:1. for ligation
    2. Use only the correct conc. of ligated mix for transformation.  too
much conc. can seriously affect transformation efficiency.

Hope this helps
jayakumar

----- Original Message -----
From: Chunxin Wang <cxwang at wam.umd.edu>
To: <methods at hgmp.mrc.ac.uk>
Sent: Tuesday, January 11, 2000 1:55 AM
Subject: CIP is the problem?


> Hi, all:
>
> I failed to make a cosmid library from a BAC clone. would you give me some
> suggestion?
>
> To subclone the BAC of my interest, I tried to construct a cosmid
> library. The vector used is pCLD04541 and clonging site is Cla I. The BAC
> DNA was partially digested by Taq I, then the 20-25kb fragments was
> recovered from the agarose gel and ligated into the above vector. 3 ul of
> ligation was used for packaging by Gigapck III XL Packaging Extract
> (Stratagene)
>
> Unfortunately, I did three times and failed to get even one clone. The
> lambda DNA control shows that the packaging extract is no problem. Then I
> used the ligation products of a YAC clone(which has been
> successfully packaged) as another control and it works very well, implying
> that my handling for package is ok. Therefore, the only thing should be
> suspicious is the quanlity of the ligated DNA.
>
> To purify the vector DNA, I used the agarose gel to recover the digested
> vector and then purify with the Bio-Rad Prep-A-Gene kit. It still didn't
> work and I always got nothing, zero colony even titering without dilution.
>
> Then someone suggested the excess CIP might be the problem because I used
> one ul of the undiluted CIP(10u/ul) from Biolabs for dephosphorylation. I
> don't believe it because the CIP was finally inactivated by heat and
> extracted by phenol. It should have nothing to do with the packaging.
>
> Any suggestion is higly appreciated.
>
> Black
>
> ---
>

---




More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net