Emergent-----Cloning problem

Wolfgang Schechinger hubahopp at gmx.de
Sat Nov 23 16:34:07 EST 2002


At 10:26 AM 2002/11/22 -0800, Haojiang wrote:

>Thank you for the response. What I did is 1. Use pBSK+ to insert the
>first clone fragment(three KB, all inserted fragment will be from
>mouse). 

>Then destroy the NotI site by NotI digestion, blunt it and
>religate. Then after Xho I digestion, I am trying to insert the second
>XhoI fragment (4.7KB). 

>The insert were purified by Qiagen Gel
>purification kit(then by QiaQuick PCR purification kit, because the
>one the one purified by Qiagen Gel purification kit didn't work very
>well). 

XhoI actually is nice for cloning, at least in my experience (HindIII/XhoI
is my favourite combination). Since your insert is pretty big, be sure to
adapt the ratio of vector and insert (normally it's given for 1kb insert
and 4kb vector), better run several ratios like 1:5, 1:1 and 5:1 (just in
case). In case of doubt, use higher overall dilutions.

The vector (6.2KB) was dephosphaylated by CIP then purify with
>QiaQuick PCR purification kit. 

reads OK. If you can get hold of SAP, better use that. You might include
the phosphatase directly in the digest (works in almost any buffer)

>I did the ligation with Roche quick
>ligation kit and the common NEB ligase(overnit ligation). 

Why mix the kits? Actually NEB T4 ligase works fine. Use NEB T4 buffer and
don't forget ATP solution shipped together with the kit. In case of doubt
(when age or freeze thaw cycles of ATP are high, better make or get some
fresh ATP solution. 

If you should suspect annealing problems, heat mix to 70 or 80 degC for
5min and then let cool to RT, put on ice, then add ligase. Then incubate at
16 degC overnight. Don't forget to include background controls. Don't use
all of the liagtion for transformation. 1 or 2 ul of a total volume of 10
normally are fine.
Then grow a number convenient for your benchtop centrifuge (say 12, 18 or
24) minipreps and check with XhoI for inserts.

Good luck,

Wo

>Then
>transform into Stable two competent cells. After use use probe to find
>the colonies that may have the insert, we make miniprep of those
>colonies. But they are just the same size as vector. But I didn'y try 
>to recover the insert form these construct. I will try.
>
>Haojiang
>
>

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