Ligation Failed, too little Vector???
Posted 20 July 2009 - 10:26 PM
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
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!!??
Posted 20 July 2009 - 11:34 PM
Posted 20 July 2009 - 11:59 PM
Posted 21 July 2009 - 01:16 AM
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,
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?
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
Posted 21 July 2009 - 01:22 AM
Posted 21 July 2009 - 07:04 AM
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.