5 replies to this topic

[hester]

  I am subcloning for my second time ever. I have a Vector that is 4.0kb  and Insert that is 1.35 kb they have both been sequentially digested with Age I and Acc65I, gel extracted , phenol etoh purified and checked on agarose gel at each step. I have tried and failed ligation 3 times. I estimate the Vector concentration is 8ng/ul. I started with 6ug but phenol and gel extraction and ciap dephosphorylation spent the lot of it. I have been trying several molar ratios.

1:1, 1:3, 1:5, 1:10
For example, I calculated

8ng V x 1.35kb I  =  10.8    / 4.04kb V =    2.67  ng I

1:1 = 2.67ng I -  8ng V        
1:3 = 0.89ng I -  8ng V

Insert = 8ng/ul so I used 0.33ul I for 1ul V (1:1 condition)
Since Takara recommends aroound 5ul total
1ul I
3ul  V
1ul TE
I got one clone that was exactly right except for one nucleotide in the third AA of my insert

So I started over and re-ligated, re plated, included control of cut vector without insert (~ 4 colonies)

Uncut vector (loads of colonies)

So I know the procedure is working, the problem has to be in ligation molar ratio, right?

and the various molar ratios gave plenty of colonies but miniprep and RE digestion showed all 23 colonies I isolated not including the insert. most of the resulting clones looked identical to Vector.

Do you think I need more vector and should start over???

Any ideas would be great!!??

Thanks

[jiajia1987]

I am not very sure about your molar ratios. For me, when I use 1:3, it means 1:3 ratio of vector to inserts and not the other way round. We do it this way here because we want all the vector to be saturated with the inserts.

[LordPhantom]

Since you have a fairly large insert compared to you vector, I would suggest using more insert as jiajia1987 pointed out. Also, did you try a positive control to check if your vector and protocol is working properly?

[hester]

LordPhantom, on Jul 21 2009, 04:59 PM, said:

Since you have a fairly large insert compared to you vector, I would suggest using more insert as jiajia1987 pointed out. Also, did you try a positive control to check if your vector and protocol is working properly?

Yes The positive control I used was an uncut version of my vector, it resulted in hundreds of colonies so, that's why it seems the problem is in ligation ratio. I believe. So, I will try using more insert next,

Do you think the amount of Vector I have is enough? to proceed to another ligation?
I think usually people are using ~50 ng of Vector, Where as I have so little and the ligation limits the volume.

I will try again

Thank you for your help

[jiajia1987]

I once had very few vector left and the normal ligation volume that I made used to be 10uL. But when I had the problem of not having enough vectors, I changed my total ligation volume to 5uL (i basically halved the volume of everything) and transformed everything into 50uL of competent cells and plated it. I hope this helps.

[tfitzwater]

8 ng of a 4 kb vector = 3.077 fmoles.
2.67 ng of a 1.35 kb insert = 3.043 fmoles.
This is the correct ratio.

I routinely perform blunt end ligations with 0.5 fmoles of vector in a 5 uL ligation reaction which for my vector = 1 ng/uL.  1 uL of ligation reaction is used to transform 25 uL of chemically competent cells.  I dilute the reaction 27-fold with SOC and plate 30 uL to generate an average of 300 colonies/plate.  

I would recommend a 9-fold dilution and plating 100 uL of cells for those with less experience.