Help - problems ligating two large framents - Ligation of two large fragments (Sep/02/2008 )

Hello everyone, I can use some help.

I have problems with the following ligation

Insert 7873bp
Backbone 9564bp

The backbone has been dephosphorelated. I set up a 1:1 molar ratio ligation using 50ng of backbone. I used Promega T4 ligase and incubated over night at 15C and another reaction at 4C. I received 13 colonies from the 4C ligation and 19 from the 15C ligation. I screened them by PCR and none had the insert.

I am about to set up another set of ligations using 3:1 and 6:1 molar ratio with the same ligase at 4C and 15C. I wanted to get some input from people here. Any suggestions?

I need help this is the smallest of 3 vectors I am making, the other two will have the same insert but the backbone will be larger 11,514bp.

Any input is greatly appreciated. Thank you.

What kind of competent cells and transformation method are you using? 13 and 19 colonies aren't that many at all. You might want to consider using company made super competent cells. Transforming large plasmids is rather difficult. I would also suggest that you run a vector only control.

How is the ligase? Do you know if it works? You should check some of your ligated DNA on the gel to make sure the ligation reaction has worked.

What kind of ligation is this? What kind of restriction enzymes are in use? Could there be problems?

How long did you dephosphorylate your DNA, and how much DNA, and CIP? Might there be problems of over dephosphorylation? Dephosphorylating the vector for too long can result in damage of the DNA ends.

Could you use more vector? 50ng isn't a lot by my standards, especially since the vector is rather large.

Were the insert and vector gel purified? Could there be problem from over exposure to UV?

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As for your subsequent ligations... I don't think you should increase the insert ratio. I would keep it at ratio of 1:1 and increase the amount the vector used. At least 100ng. Once the ligation has finished, i would check to see if the ligation reaction had worked (run the ligation mix on a gel and look for high molecular weight bands). I would also do a vector alone ligation as a control.

Also you can be a bit paranoid and miniprep the colonies and test them by restriction digest. Sometime the colony PCR can fail (especially if you used too many cells). It will probably confirm the PCR results, but you never know.

The problem is usually before the ligation, not during it. What REs are you using? Can you avoid gel purification and use double cutting rather than dephosphorylation? In general, the less you do to your DNA the better. Where did the insert come from? PCR? Is this a blunt or cohesive end ligation? Tell us (much) more.

Wow, I did not realize i left so much out. Thank you for getting back to me, here is more information

1. I can not use different RE, so I am stuck with AscI. I digested 2ug of DNA (plamid cut the AscI which will be the bacbone)

2. Dephosphorolation was done the rAPid ALkaline Phosphatase (rAP) kit from Roche.
I used the digested 2ug of DNA and added 2U of the rAP, incubated at 37C for 10min (Kit recommends 1U per 1ug of DNA)

3. Did not gel purify the bacbone, i cleaned it the PCR clean up kit from Qiagen.

4. The insert was gel purified, along with other fragments for other cloning steps, so far the others have worked well. So I am thinking that this is fragment is also fine. The insert comes from another plamid and it was cut with AscI and as mentioned gel purified.

5. The Promega T4 ligase is fine, since three other ligations were done at the same time and they worked. The final plamid sizes for those were two 11.5KB and one 15.5KB. The one that did not work was the 17.5.

6. Cells are the mach1 cells from Invitrogen. I have ordered XL10-Gold from stratagene, they are supposed to work well with larger plasmid, I hope this was the problem.

7. The colony PCR: I think its fine since these primers were used for the 15.5Kb ligation and they showed correct results. If these primers had not worked on the other ligation, i would have done what "perneseblue" recommends and minipreped and digested my PCR negative colonies.


I have all the questions you have asked, thank you.

Thank you for the input on the molar ratio. I will keep the 1:1 molar ratio and increase the amount of vector. Maybe do 100ng and 200ng.
I will run some on the gel to see if the ligation worked. I hope the ligation is working. I will also transform into the Mach1 and the XL10-gold see if the cells make a difference.

Any other ideas, questions? Thank you.