Double digestion and ligation problem - (May/17/2004 )

Hi

I`m trying to set up ligation but I really have problem with it now. My expressing vector is pCP60 derived from pBin19 and it`s 12,5 kb. The insert is about 900 bp (arabidopsis t. gene). he restriction e. were BamH1 and Sac1.

so, if anybody have a troubleshooting offer, I appreciate her/his help.

cheers,
mely

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I'm facing some ligation problem too...

First of all,
i double digest my insert; in fact 4 of them(600kb, 700kb, 800kb and 1 2.4kb) with this BamHi and KpnI from my pDrive(QIAGEN PCR clonig kit). I also double digest my PXJHA vectors with the same double digest.

Then i try to ligate them in 4 different reactions but for 3 times, no colonies ever grow at all... I wonder whats wrong.

my conditions:
1.5ul of vectors
1ul of T4 ligase
1.5ul of ligase buffer
5ul of insert
6ul of water (i used autoclaved milliQ water)
total reaction volume is 15ul
Then incubate at 16oC overnight.


I also try using another vector;pEGFP-C1. I double digest it and my insert with BAmHI and XbaI. BamHI is a must while XbaI is the only cut site available. However, THere is a methylation at this XbaI cut site which mean it will not be cut at all...(am i rite?)
So after i double digest them , surprisingly, the vector is cut so i ligate both the vector and my insert using the same conditions above.
FInal result:==> no colony growth at all..

Dear all, please help....I have been doing this for 2 weeks and still cant get it...I cant think of anything that i have done wrongly.

and my boss just keep saying 'try again', 'there's no reason why it wont work', u must have done something incorrectly', why are you so slow in producing these clones etc...

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Hi all
Iam facing a similar problem Iam trying to do a plasmid double digest with sfi1 and Not1,digestion with one of them alone is giving good results but the double digest yeilds poor or no results what can Ido

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SfiI is not an enzyme I would b comfortable working bcoz of it properties.

Could you select some other site for ur cloning. Might b better.

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I also digest my vector and insert with first the Sfi1 and then the Not1 enzyme in one reaction tube (double digestion but not at the same time since the condition of the enzyme activity are not the same) but I always get good results. So I'll suggest you elaborate on how do you prepare your double digestion reaction then we'll take it from there!

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I am doing the double digest in two separate tubes ,firstly digestion is done with sfiI then NotI using transformer buffer (containing higher concentrations of Buffer G).

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