Hi all
I would like to know whether a vector digested with blunt end cutter enzyme, be cloned with an insert which as got sticky end. To explain you in details i am cutting my vector with SmaI enzyme and I want to ligate this vector with an insert which has got EcoRI sticky ends. Will this ligation work? Please do help.

hallo buddie,

it is not so usual to ligate sticky with non-sticky ends. I suggest, to ligate some so called linker-DNA to the sicky end and digest after it with the appropriate enzyme to produce sticky ends. Finally you have two sticky ends to ligate.

I hope this helped.

some informations e.g. http://www.fermentas.com/techinfo/modifyin...kerligation.htm

if u are very particular about the sites then u should probably only use a linker to avoid degradation of the ends. otherwise it is simple to digest the ends with klenow and ligate it.
good luck!!!

if your vector cut site is blunt then you should blunt your insert as well, otherwise you cant get ligated plasmid

Hi all
thanks for your suggestions. People I've used Pfu pol for getting blunt ended insert. The problem I face here is when I use Pfu pol along with the amplicon of expected size I get several other bands of various sizes. But when I use Taq pol there is no problem.
I have to cut the vector with SmaI. So only I seeked your help whether Taq amplified insert and blunt ended vector can stick. If anyone can give me some suggestions in this matter it is highly appreciated.

Taq adds an A on the 3' overhang to 70% of the products it makes. Furthermore Taq make alot of mistakes. Taq is not the best candidate to make blunt end products. Although if your insert yeild is high you can get away with it.

You could change the PCR conditions and make it more stringent to reduced secondary band formation (ie rasing annealing temperature). Alternatively you could gel purify your DNA fragment.

Best of luck with the blunt end ligation

Hi pernese blue
I am aware we can't use Taq pol for making blunt end products. I use this for preparing inserts to clone in pGEMT easy vector. I dont have problem in Taq and pGEMT cloning. I wanted to use another vector where I have to make blunt end ligation and insert concentration too low when I use Pfu pol. I will try to follow your suggestion for reducing unwanted amplicons. Thanks. Is there any other suggestion for increasing the efficiency of blunt end ligation...........

In the specific situation of blunt end ligation, you could try using Quick ligase buffer, ie adding PEG6000 to a final concentration of 10% into the ligation mix. The ligation efficiency is improved. However the PEG has to be removed by an EtOH wash as PEG reduces transformation efficiency.

Hi blue
I've tried using your suggestion of adding PEG in ligase buffer. but this has not helped me much. I do get colonies but they are not recombinants just transformants of the vector. anyway thanks for your suggestion
then I've used a transformation buffer which includes PEG and I don think PEG will lower transformation efficiency. I've got good results with that transformation buffer.