Ligation or transformation problem - (Jul/14/2006 )

Hi,

I'm now cloning a 1.3kb fragment (from PCR) into pEGFP vector (K+). However I cannot got any colonies for several times.

I'm now describing details of my protol as follows:

I used reagents from NEB, and exactly acted by the instructions.
The DEs I used were Xho I and Nhe I, whose functioning conditions were compatible when using at the same time.
I did single digestions of plasmid to see if the Ens work, and could see the bands in gel comparing to original plasmid.
I also could see the double digested plasmid band, but because the two digestion site is close I couldn't tell difference between it and the single digested one.
After inactivation of both Ens, I did CIP to vector, then inactivated again, and purified both insert and vector DNA using the kit.
Then I ligated them. 16 centidegrees ovn.
The insert:vetor molar ratio is 4 or 8 (I made 2 trials).
The total DNA concentration of insert and vector is between 6 or 8 ng/ul.

After transformation, there's no single colony in any plate! (DH5alfa is ok when I transformed with intact plasmid)

Anybody can give some advice? I'm really going crazy now...

Regards,
Josophen

How long did u digest ur PCR and the vector ?

Normally I ligate at RT for 1hr when using NEB T4 ligase.

Which is the total amount of DNA you used for your ligation? And the trasformation efficiency of your cells? When you did your transformation with intact vector, did you use the same amount of DNA you would use for the ligation or more? And did you give the cells time to express the K resistence?

is your ligase ok?

(to check, simply try to ligate your lambda HindIII ladder back together - run on gel - one band or many?)

Thank you guys!!

scolix:
I digest both overnight.

dnafactory:
Total amount of DNA used for ligation is 6-8 ng/ul.
Trasformation efficiency is a little lower than usual in our lab.
The intact vector amount for transformation is far smaller than the amount I used for ligation.
I let cells grow in LB about 1h before plating.

aussieuk:
Ligase is new and ok.

So anything can be wrong?

I looked up the map for pEGFP and it appears that nheI and xhoI do not cut within this vector. I couldn't find the specific (K+) plasmid you indicated.

http://www.clontech.com/clontech/techinfo/...DF/pEGFPdig.pdf

However, if you used the above plasmid you may only get a lawn after plating your ligations. Is that what you got or did you get nothing. "there's no single colony in any plate!" - does this mean you got no colonies or 'no single colonies', ie. a lawn?

If you are using the vector without the xho/nhe you will need to pick different sites.

I would suggest to digest ur insert and vector during the day (few hours) instead of overnight as sometimes the ends can b over digested and u will not get any colonies.

Also b sure abt the sites as vasussci suggested.

Erm, I not sure if ur ligation and transformation has worked but I just want to ask if u added BSA when u did the digestion? cos I think both Xho I and Nhe I requires BSA. If u have added, then forget wat I have just said, So sorry, if I wasnt of much help!

Good luck to you! Don give up so soon!!must try your best!

Thank you again!

vasussci:
Well, the vector I'm using is pEGFP-N1, which is 'discontinued' accroding to Clontech's website. See here. I'm sorry to omit the full name.
So there're digestion sites in it. And 'no single colony' means no colony at all...

scolix:
I'll try that!

lusi:
I've added BSA into the reaction...

Any other suggestions?