Weird result (ligation) - (Mar/03/2007 )
im having a series of failures in ligating an 800bp insert into my low copy 8kb vector. Both the vector and insert was sequentially digested by Kpn1 and BamHI, 2-3 hours digestion for each. The vector is temperature sensitive (low copy at 37C , high copy at 42). My vector does not contain lacZ so no blue/white screening was made.
Due to my ligation failures i made 5 sets of ligation to check where the problem is.
Set 1- 1ul vector only (cut)
Set 2- 1 ul vector (cut) plus ligase, buffer, distilled water
Set 3- 1 ul uncut vector
Set 4- 1:1 ratio of vector to insert ligation
Set 5- 1:3 ratio of vector to insert ligation
Ligation O/N at 16C
The results
1. more than 300 colonies
2. none
3. lawn colony (well competent cells)
4. 10 colonies
5. 15 colonies
The problem is the results doesnt make sense. I thought that the colonies from set 1 is due to my vector not being digested well. However, i wonder why i have no colonies on set 2. My competent cells is ok as based on set 3. I havent checked Set 4 and 5 for insert presence but i really doubt it bec of the results of my set 1 and set 2.
Can anyone explain to me where the problem is and how can this be happening? Cloning gurus pls help. Thanks
While it is surprising that there are zero colonies, you would not expect many, since the vector has been cut with two different enzymes. Religation between the incompatible ends is rare. I would be checking the colonies on the insert containing plates with reasonable chances of having the correct construct.
I would suggest you check some of the colonies in set 4 and 5 for insert. Cloning always doesnot makes sense.
I am not sure why you have more than 300 colonies in set1. But if you keep set 1 aside, others makes sense, doesn't it ?
You have used the same vector, which comes form the same tube, for all of them. So without ligase if you get 300 or more colonies, you should get at least similar numbers with ligase. Well, dont worry too much, and check the colonies for inserts and I would guess you should have the insert.
i used to think too hard to explain cloning, but then I gave up trying to sort it.
Good Luck !!!
I am not sure why you have more than 300 colonies in set1. But if you keep set 1 aside, others makes sense, doesn't it ?
You have used the same vector, which comes form the same tube, for all of them. So without ligase if you get 300 or more colonies, you should get at least similar numbers with ligase. Well, dont worry too much, and check the colonies for inserts and I would guess you should have the insert.
i used to think too hard to explain cloning, but then I gave up trying to sort it.
Good Luck !!!
Thanks phage434 and scolix. Im doing the checking now and il update as soon as possible
more power something in the ligase or the buffer could be inhibiting the transformation...?
too bad..i have no transformants 
ok, well the fact that you have 300 transformants from your plate with 'cut' vector, suggests to me that the vector isn't digesting properly. What vector is it? Do you have the same vector with another insert available?
hi scrat. my vector is POU254 derived vector, where i deleted the lacZYA then self ligate it . its a low copy temperature sensitive vector. Ive never used that vector in any cloning except for this one. and its a failure
pls advise. thanks hmmm, well I can think of a couple of things that might be happening.
Firstly, if the vector is not digesting properly, are the enzymes old? Is your BSA still OK? Are the enzyme sites very close to each other on the vector? (I'm not sure if this vector has a multiple cloning site).
This may be a long shot, but does your vector have priming sites flanking where the lacZYA used to be, eg M13 or SP6? You could do a PCR on your purified cut vector, if the cut sites are between the 2 primers you should get no product.
It's also possible that you have contamination with another vector somewhere. You said you get no inserts from your two ligation plates, are you screening by digestion or PCR? Does it look like you are just getting empty plasmid instead?
Firstly, if the vector is not digesting properly, are the enzymes old? Is your BSA still OK? Are the enzyme sites very close to each other on the vector? (I'm not sure if this vector has a multiple cloning site).
This may be a long shot, but does your vector have priming sites flanking where the lacZYA used to be, eg M13 or SP6? You could do a PCR on your purified cut vector, if the cut sites are between the 2 primers you should get no product.
It's also possible that you have contamination with another vector somewhere. You said you get no inserts from your two ligation plates, are you screening by digestion or PCR? Does it look like you are just getting empty plasmid instead?
hi scrat..the enzymes are new, as well as the BSA. However my enzyme sites are really close to each other. Separated by 2 base pairs. Can you pls explain to me how can that be a probem?im starting to think thats the main reason but i dont know how to explain it to myself. My other option is to design a new set of primers, however i want to make sure that the vector itself is not really the problem, except for that 2 RE's that are very close to each other.The vector have MCS. Im screening by digestion, since i dont have primers for the vector, though im planning to design one. Yes, im getting plasmid instead with no inserts.