Cloning Help!! - It is getting very annoying... (Jul/29/2010 )

Hey, I am currently cloning a 1.8 kb insert into a 8kb vector with AMP marker. Both are digested with SacI and XbalI at 37, and the vector is CIPed for 1 hr at 37. I did my ligation using T4 ligase for 30 mins at ROOM temperature, then transformation. However, I see little to no colonies in both actual and negative control. I counted the colonies and they are approximately the same number. Here is my protocol.

The insert is PCR amplified to add a XbaI cut site.

Restriction Digest

Insert
5 ul Neb 4
5 ul 10x BSA
1ul of insert
0.5 ul sacI and xbaI
38 ul of ddH2O

Heat inactivated at 65 for 20 mins then on ICE

Vector
5ul Neb 4
5ul 10x BSA
1ul of insert
0.5 ul SacI and xbalI
38 ul of ddH2O

Heat inactivated at 65 for 20 mins

on ice for 10 mins

CIP the vector for 1 Hr at 37


After gel purification
=29.1 ng/ul

=8.1 ng/ul

Ligation 20 ul Rxn

actual
2ul t4 ligase buffer
2ul t4 ligase
4ul of vector
12ul of insert

control
2ul t4 ligase buffer
2ul t4 ligase
4ul of vector
12ul of ddh2o

ligation at room temperature for 30 mins then heat shock transformation

What am I doing wrong here? I have discussed it with my lab mate and she said my vector concentration is too low; it needs to be at around 50 ng. I am currently doing another digest with 10 ul of vector this time. I am getting really ticked off at failing, I am about to throw my stuff at my prof.. help please >.<

I would use much more than 1uL of insert and vector in your initial digests, try bumping that up to 20uL or even more for your insert, the more insert the better.

I would also digest for more than 1 hour @ 37 degrees, if xbaI and sacI are both compatible for double digest, how long they last and star activity is favourable I would go for 4-8 hours.

Then instead of heat inactivation I would use a spin column to purify the digested DNA.

also you dont have to CIP treat your vector unless this is a blunt end ligation, if its cohesive (sticky) ends CIPing your vector will decrease efficiency... if you must CIP treat your vector then kinase treat your insert as well - so you know you have phosphates on insert and none on vector.

and ligate for more than 1 hr at room temp, i usually do overnight @ 16 degrees then heat inactivate the ligase since it can decrease efficiency of the transformation.

hope this helps, my guess would be the CIP treatment is whats going wrong.

not gonna load it on a gel? I am not very confident with my digest, even tho it works everytime...

I thought CIPing prevents re-ligation? Shouldn't it be a must?

yea you can run digests on a gel to check they went ok.

the linear vector can only self-ligate efficiently if the two ends are blunt or compatible... are xbaI and sacI cuts blunt or compatible? Im pretty sure they are'nt. Do some reading up on ligations, starting here: http://en.wikipedia.org/wiki/DNA_end

or you could be scientific about it and do a CIP and non CIP treated vector and see which one works, if its the non-CIP treated vector then you owe me a beer

cluelessstudent on Fri Jul 30 04:18:37 2010 said:

Yes and no.
1-Overdephosphorylating with CIP has a nasty tendency of causing damage to the end of a DNA fragment, rendering the molecule unligatable. To test if your vector is still ligatable, treat the vector with PNK + Ligase and see if you can get any colonies (if you can spare the competent cell). Alternative you could run the ligation mix on a gel and see if you can observed high molecular weight DNA bands... indicating ligation has occured.

I have the feeling that the vector has been over dephosphorylated and maybe unligatable. Please check.

2- The vector has been cut by XbaI and SacI, if the digest had gone to completion, the end of the vector are now non-compatible. The vector should not be able to self ligate. As a result some people leave out the dephos step when the ends of the vector are none compatible.

How long did you do the restriction digest for? And how much DNA was cut?

perneseblue on Fri Jul 30 05:11:22 2010 said:

1 hr at 37 degrees, using 1ul of 300 ngl/ul of DNA. It should be enough, I saw a really intense band in the gel

XbaI is sensitive to dam-methylation. Which E. coli strain did you use to prepare your vector? Should be dam-negative for your purposes. Otherwise the digestion will not work. Did you check the vector-digestion by gel electrophoresis?

My quick comments:

The NEB BSA is at 100x, not 10x. You are adding 10x too much. Probably this is not a problem, but should not be routine.

I would definitely try this reaction without the CIP treatment, as suggested by Pernesseblue.

The PNK treatment of your insert is ridiculous -- it already has the phosphates.

Heat killing works fine, there is no reason to do the column prep.

XbaI is not sensitive to DAM methylation unless there is a leading GA before the TCTAGA site, or a trailing TC after the site. You can check the vector, but almost certainly the vector has been designed to avoid these, and there is no need for a dam- strain.

The PCR product will have no digestion issues, but you should check and verify that there is a 5' overhang of the XbaI site on your pCR primer.

I would cut a little more of the DNA, but probably not as much as 20 ul out of a 50 ul reaction, to avoid inhibitor issues.

I agree with the concerns voiced about CIP. I wouldn't do it at all if it's not needed, and it sounds like it isn't. If you do CIP, use much less and for far shorter amount of time -- I typically use 1/10th to 1/5th the amount recomended and allow the reaction to proceed for no more than five minutes...

it's funny tho, many of my lab workers said that without cip, the sticky ends somehow get back together even when they are non-compatible with 1 another... any1 know why? It seems those restriction sequence are not that "specific"...