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Strange results after transformation - (Jun/27/2005 )

I ligated a 9kb fragment, on both sides cut with NheI, into a dephosphorylated 2.6 kb vector single digested with NheI and did a transformation using XL10 Gold competent cells.
I would expect a 11.6 kb vector carrying the fragment in two possible orientations.
This are the results of my transformation:

ratio fragment/vector......... DNA used for transformation.............. number of colonies

1:3 ligated ...........(15 ng fragment, 14ng vector) .................. 32
background control ligated ........(just 14ng vector) ..........40
1:12 ligated ........(15 ng fragment, 54 ng vector) .......7
background control ligated ..........(54 ng vector) ............54
vector dephosphorylated unligated ........(14ng) .........70
pUC control .........(0.02 ng) ................> 1500

the transformation was performed in 50ul of competent cells.
In the controls I theoretically shouldn't expect anything.
I didn't check the colonies, but due to the transformation results listed above its very unlikely that there are positives)
I used an extra long incubation time for the CIP dephosphorylation of the vector to get sure that everything is dephosphorylated (and to minimize background) .
The results suggest that the the initial restriction digest of the vector was not complete and I still carry the undigested vector with me (actually I checked this on a gel...but I just could see linearized plasmid after NheI digest). But even after a partial digest and successful ligation I should expect a difference between control and insert/vector ligation....
Since I did the experiment in many different ways....I'm really desperate!!
Please help!

-prize laurate-

when u said u used an extra long incubation time for the CIP dephosphorylation of the vector to get sure that everything is dephosphorylated, i was thinking long time dephosphorylation could do harms to the insert. sometimes, there will be nucleotides lost at the ends of your insert. So long time is not always good.


How did you purify your insert (the 9kB beast)?

Sounds like something in there is inhibiting the ligation.


I got the 9kb beast out after a single restriction digest with NheI, run it on a gel and cut out the 9kb band. I purified the beast with Qiagen gel extraction kit.
pBluescribt, do you think the salt in the buffers provided with the kit could inhibit the ligation? Do you think especially big fragments could be affected?

Hsm142, thanks for your hint...I know that CIP is a nasty enzyme...with long I meant 90 minutes insted of the recommended 60min. I've got the feeling that a too long incubation is not the reason. I think my problem is the high background I couldn't get rid off and/or that the ligation didn't work.
Any further suggestions?
Thanks so much for your help!

-prize laurate-

prize l. I have been using many quiagen gel purification kits recently and none of them have been inhibited during ligation/transformation, so I don't think anything is wrong there, unless you got a bad kit. Are you eluting from the column with the EB buffer? I always do.

Could your ligase be bad? Do you heat your ligase reaction to 70C when you are done ligating?

Jeeze, I always hate it when backround colony numbers exceed recombinant colony numbers.

Of course you are trying to force a 9kb beast into a itty-bitty vector... can you change to a different vector?

How about retrying the ligation by keeping vector mass the same and varying insert mass? How about this.... digest out your 9kb beast and CIAP treat that instead of your vector.... see what happens.