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Ligation/Transformation Problems 9I'm not sure which!) - (May/05/2009 )

Hello,

I've been trying to ligate two inserts (1800bp and 900bp) into separate EGFP-C2 vectors and I appear to have the ligation working but I don't have too much success when it comes to the transformation. I do 20ul ligation reactions using 1ul T4 DNA ligase from Invitrogen and 10ul insert+4ul vector. Inserts and vector are digested with EcoRI and SalI. Vector is gel extracted with Qiagen gel extraction kit while the insert is purified after digestion with PCR purification kit.

After purification, I have ~2-3ng/ul vector which I can't see when I run on a gel and -10-15ng/ul insert which I can see on a gel. I do 16C o/n ligation and then run 5ul on a gel at which point I see one single band along with 2-3 bands above this so I am presuming my ligation has worked. After each ligation I get the same pattern on a gel so I'm doubtful that I'm getting concatamerisation each time.

However after carrying out a transformation I have no colonies. I'm using the subcloning efficiency DH5a cells and the supplied positive control gave colonies as did the undigested EGFP-C2 vector (few colonies though).

I'm now confused as I'm almost certain my ligation works since I get the same result on the gel each time so I'm beginnig to wonder if the transformation is the problem. I use 30ul cells and have tried 1ul, 5ul and 10ul of the ligation reaction. I incubate on ice for 30min, heat shock at 42C for 90s cool for a few min onice, followed by 1hr at 37C and 225rpm (900ul LB broth added to the cells).

I'm relatively new to cloning so if anyone has any suggestions, I would really appreciate it!!!

smr86

PS Attached is an image of one of my gels lanes 5-8 contain the ligation product
Attached Image

-smr86-

Hi smr,
maybe try using 50l DH5a cells with 4-5l ligation mix. You could also heat-inactivate your T4-ligase prior to transformation. Additionally, try a heat-shock of 45 secs + 2min on ice, than add 250l of antibiotic free LB prewarmed to 37C/42C.

On the other hand, 2-3ng/l backbone is not too much. How much vector (EGFP) do you digest for gel purification(try 1-2g)? Is your vector completely digested(try o/n digest)?

As ratio vector : insert you can use 1:4 to 1:10 (Ratio1:Ratio2)

If you consider backbone and insert size and concentration you get:

l2 = (l1 x Ratio2) * (Size2 x Conc1)
---------------------------------------
(Size1 x Conc2) * Ratio1


where 1 is backbone and 2 insert

good luck
Fred
Attached File

-fred_27-

Hi Fred,

Thanks for your suggestions, I have tried 50ul cells for the transformation and didn't get any colonies. I'll give it a go inactivating the ligase prior to transformation and see how that goes.

I'm very suspicious about my vector conc though. I digest 1ug for 3hr and then gel purify it but I just don't get a very high conc. at all. I've tried a fresh kit from Qiagen and that didn't help. My supervisor did suggest a phenol chloroform extraction but I heard the phenol could disrupt the ligase activity so I'm reluctant to try it.

Hopefully I'll get it to work soon!!

-smr86-

Hi smr,
there are some ways to increase your backbone conc.
a) digest 2 to 4 g DNA, maybe o/n
b) elute DNA with smaller volumes (eg 2x20l is ok / 1x20l)
c) prewarm elution buffer to 60C

Fred
Attached File

-fred_27-