12 replies to this topic

[pragathi]

I am trying to clone a 800bp insert into a 11kb vector at BamH1 site on both sides. After restriction digestion I do a phosphatase treatment and gel purify the vector before ligation. I have tried several ways of ligation...   molar ratio- 3:1, 5:1, 10:1 ratios of vector:insert, incubation at 16 degree C 4 hours, 4 degree C overnight, 2 hours room temperature incubation but am unsuccessful   .

I use invitrogen one shot high effeciency chemically competant cell. I have even checked the transformation effeciency by positive control with the uncut vector and got good no. of colonies. so I am sure the problem is in ligation. I have tried both invitrogen and fisher sci. T4 DNA ligase enzymes.

Please suggest me anything else I can try.
Thankyou in advance

Edited by pragathi, 28 June 2010 - 06:52 AM.

[pito]

Have you tried an extra purification after gelpurification?
(extra purificiation: ethanolprecipitation..)

this might be needed.

[ram]

Are you sure that your restriction enzyme, phosphatase and ligase are working? You can try using different vials than you used before.

[pragathi]

Thanks.
I have done once extra purification using DNA clean and concentrator kit from Qiagen after gel purification using gel purification kit.

[pragathi]

Thanks.
I have done once extra purification using DNA clean and concentrator kit from Qiagen after gel purification using gel purification kit.

and I have checked the ligase activity by ligating lambda DNA. It works. regarding the phosphatase-- there is no self ligation and restriction enzymes are working well as I can see good bands for vector and insert on the gel.

[perneseblue]

I would also suggest that you check to make sure that the dephosphorylated vector is ligatable

If you are dephosporylating with CIP be careful. Over dephosphorylation can render your vector unligatable.

Treat some dephosphorylated vector with PNK and see if the vector will religate.

[pragathi]

Thankyou, will try this treatment..

[swanny]

Quote

have tried several ways of ligation...   molar ratio- 3:1, 5:1, 10:1 ratios of vector:insert

Did you actually do it this way, or did you do it the reverse, i.e., 3:1, etc insert:vector ration?

[pito]

swanny, on Jul 2 2010, 07:26 AM, said:

Quote

have tried several ways of ligation...   molar ratio- 3:1, 5:1, 10:1 ratios of vector:insert

Did you actually do it this way, or did you do it the reverse, i.e., 3:1, etc insert:vector ration?

What would you suggest?

I always use more insert then vector.

So maybe that might be his problem. I must have missed the fact that he used more vector then insert.

[ram]

pito, on Jul 2 2010, 02:31 AM, said:

swanny, on Jul 2 2010, 07:26 AM, said:

Quote

have tried several ways of ligation...   molar ratio- 3:1, 5:1, 10:1 ratios of vector:insert

Did you actually do it this way, or did you do it the reverse, i.e., 3:1, etc insert:vector ration?

What would you suggest?

I always use more insert then vector.

So maybe that might be his problem. I must have missed the fact that he used more vector then insert.

I guess the optimum molar ratio would obviously be 1:1 (equimolar). So, for example if your vector is 3 times larger than the insert, you should use 3 times higher amount (for example, in nanograms, NOT molar) of vector than the insert to get equimolar quantities of both

[perneseblue]

I agree.  I too believe that the 1:1 mol ratio of is best. Equal number of molecules.

[josse]

What do you mean with 1:1 mol ratio?

You mean you need to have the same amount of mols of the vector and the insert?

And how do you calculate how much mole of each you have?
Normally you only know how much bases it is?

[ram]

josse, on 31 July 2010 - 07:36 AM, said:

What do you mean with 1:1 mol ratio?

You mean you need to have the same amount of mols of the vector and the insert?

And how do you calculate how much mole of each you have?
Normally you only know how much bases it is?

Yes!  1:1 mol ratio means same number of moles (and hence molecules) of insert and vector and it is preferred because 1 molecule of insert ligates to 1 molecule of vector. You need not know absolute number of moles of each, rather you are interested only in their ratio and while doing so it has to be assumed that the molecular weight of a fixed length of DNA having any composition would be the same (same GC content).
If length (number of bases) of insert is 'x' and that of vector is '3x' (3 times larger than insert) than a particular weight (for example, in nanograms) of insert will have 3 times the number of moles in the same weight of vector. Although ready-made calculators are available at websites like this, I will recommend to get your moles, molar and molecular biological concepts cleared if it is still difficult for you.
Good luck!