klenowed PCR fragment into EcoRV digested bluescript - blunt end ligation proble - (Dec/19/2006 )

hello,

ive been trying for a while to get this blunt ligation to work.....please help!

insert: 450bp PCR product -> prot k treated -> gel purified -> klenow treated -> kinase treated

backbone: EcoRV digested pBluescript -> gel purified -> CIP treated -> phenol:chloroform extracted

I always use a backbone alone ligation control which gives me 1-2 blue colonies so i guess the CIP treatment is working ok...The ligation mixture shows up 1-2 blue colonies as well. No white colonies.......I have tried a 1:3 and a 1:5 molar ratio of backbone : insert.

I use roche T4 DNA ligase....ligation reactions are in 20microlitre volumes with about 100ng of backbone and approx. 10 ng(1:1), 30ng (1:3) and 50 ng (1:5) of insert. I use 5 microlitres of the ligation mix to transform into DH5alpha.

I have tried the ligation without phosphorylating the insert but without any luck. I will be transforming a 1:10 ligation today (fingers crossed!!)...

should i try ligating in a 10 microlitre volume?
maybe i should add PEG to the ligation?

any suggestions will be most appreciated...

thanks very much!
fyre

Using 1-2.5 ul of ligation mixture for transformation is sufficient.

good luck !!!

PEG could help.

How long are you ligating for? An overnight ligation at 16 Celsius might help. Could even drop the tempearture to 4 Celsius (that helps sometimes)

Have you considered a different cloning strategy. Perhaps engineering restriction sites into your PCR product and then proceed by a sticking end ligation.

You could leave your vector phophorylated. And do the ligation from there, using the Xgal/colour test to screen for insertion. There is the possibilty that your kinase step has not worked.

There is a very strong possibility of concatemers in your current cloning strategy. So do check for multiple insertion events.

thanks for your comments guys!..

i forgot to mention that i had used the exact same method to clone a different gene into pBluescript a few months back....dunno why this time it isnt working....

scolix, I usually use about 2 ul of my ligation mix to transform....i used more this time since its a blunt ligation and i thought i'll chuck in a bit more.....i transformed using 3ul from my 1:10 ligation today.....prayers are on!.....

perneseblue, i ligate overnight at RT (about 16-20 deg C)...i havent tried ligating at 4 deg C....will give that a shot....
i thought about ordering fresh primers incorporating restriction sites....will do that if the next couple of trials dont work....i shouldve done that in the first place!....<dumbass>....
im not too sure about the kinase myself.....its a bit old....could have expired.....

thanks again!...

fyre

white colonies!!!....i have white colonies!!....wooohooo!!!....<does a little jig>.......

got 7 white colonies....but a gazillion blue ones....and my vector-alone control has 2 blue ones....crazy!....setting up minicultures now....hope to have good news tomorrow!.....

fyre

finally!!!....it worked......

fyre