Ligation trouble continues - subcloning ligation failure (Aug/05/2009 )

I am currently subcloning a
1.35 kb fragment (pcr product with RE' sites added ( AgeI and Acc65I) into a
Vector that is 4.04kb long and was digested, phenol+etoh purified and then gel extracted.

I have tried ligation 4 times now.
I have re-made the vector (originally 4.8kb) because I was afraid that Some DNA contamination might have occurred from inefficient sequential digestion. The two restriction sites I am using are not compatible for a double digest. So I am digesting the one which requires a lower salt content first and then adjusting the salt content and adding the 2nd RE.

I was able to see the fragment of 780bp cut out of the vector resulting from the digestion.

My plates are in several molar ratios (V : I) Uncut vector only, Cut vector only, 1:1, 1:3, 1:5 AND 1:10
My resulting colonies were tons, ~20, ~60, ~60, ~35, ~35

But when I miniprep 30 of them I found vector only

So today I am beginning with preparing the insert again with a new round of PCR. This time I will add purification with phenol steps, following PCR and in between digests (I previously didn't do this)

This should be a straight forward sticky end ligation! If this doesn't work I may have to blunt end ligate the pcr product to another vector, clone that and than cut the insert out from there and try ligating. But that seems like so many extra steps and I already feel like this has taken way too long. Please if anyone has some ideas. I am more than happy to answer any specific questions you may have

I recommend doing less, not more, to your DNA.

Before you start, check:
* adequate overhangs at the 5' end of the pcr primers
* adequate space between cut sites on the vector
* no dcm methylation issues with the vector sequence preventing acc65I cutting

Do a PCR, clean up the pcr reaction with a column (check for a correct sized band, concentration). Digest both the vector DNA and the purified PCR product with the two enzymes. Do as little as possible. Adding salt to adjust concentrations sounds right, but you could also try a double digest in buffer 2. Do the digestions in decent volumes and low enough concentrations. 50 ul reactions with 1 ug of DNA is a good spot.

Heat kill the enzymes (no purification).

Mix equimolar amounts with about 20 ng of vector in a 10 ul ligation reaction with T4 DNA ligase. Use the normal ligation buffer, not the quick ligase. Ligate for 30 minutes at room temperature. Make sure the ligation buffer is fresh (it can go bad on freeze/thaw cycles). If uncertain, add some ATP.

Do a similar ligation with vector only DNA.

Transform 2 ul of each ligation reaction into 50 ul of competent cells. Also do a transformation control with 1 ul of pUC19 DNA diluted to 10 pg/ul. Your control should show at least 10^7 cfu/ug of pUC19.

Compare colony counts on the two ligation plates. The insert plate should have more transformants, and you should pick some and evaluate. I would use colony PCR, but you can do minipreps.

If you have too many transformants on the vector only plate, think about SAP or antarctic phosphatase treatment of the vector, but I would try to avoid it. If you do it, do it with minimal units and minimal time.

Hi hester!

I agree with phage434

I think you are loosing your DNA when you are going through unnecessary purification steps
For example, if you are planning to do gel purification down the road, you don't need to purify your vector with phenol-ethanol.

My suggestion: Run 5 ul of purified vector and insert (from the same tubes you use for ligation) on an agarose gel.
If you can see your bands clearly - proceed with ligation. If you can't see your bands - that's is the first reason for your ligation not to work.

Best,

Andriy

www.simplycloning.com

hester on Aug 5 2009, 12:43 PM said:

How is that possible? The vector should be linearised and incapable of self-ligating, right? I'm pretty new to this, so maybe I'm missing something, but it suggests to me that either

1) you extracted the wrong band from the gel (did you take an uncut control for comparison?)
2) if the right band was extracted, then self-ligation must have occured? In which case, could you try CIPing the vector before ligation?

I'm assuming that linearised vector cannot be repaired and replicated by bacteria? I think that's the case.

I also agree that purification of DNA prior to purification by gel extraction is unecessary. Just go straight from restriction to gel-purification? I did a 16-hr restriction, and didn't bother with heat inactivation before gel-purification but you might, if the restriction is of shorter duration?

seanspotatobusiness on Aug 8 2009, 04:25 PM said:

Thanks for the response!

I agree that it should be incapable of self ligating, I absolutely took the right band and dephosphorylated the vector before gel extraction too. But I noticed that sometimes a small amount of undigested vector can remain and you can not distinguish that small amt on the agarose gel. That's at least what my advisor said to me.

I re-prepared the Insert yesterday and ligated today, because I think maybe the insert wasn't digested well. The insert has the RE sites attached at the ends with just 5 extra nucleotides, so the Agarose gel can't confirm that the digest is complete.

Previously I did not include a clean up step after PCR. However this time I have included it and followed phage434 advice.

So, I am crossing my fingers tonight that this works.

If it doesn't I will clone the insert by blunt end into Topo PCR cloning kit or something similar and sequence the insert and then digest it from that vector and ligate it to the vector I want it to be in.

I should mention that I previously obtained a good clone except for one nucleotide in the third AA of the ORF (which was apart of the PCR primer I ordered and double checked again and again), it was a substitution - C became T. I think that it was a really odd thing to have wrong...

I will say that for all the anger and frustration cloning is causing me, I feel like I have definitely learned a lot about the procedures!

Clean up after PCR is critical for cloning applications. While the RE enzymes will gladly cut in PCR buffer, the PCR enzymes will extend the cut 5' overhang ends, destroying their ability to ligate.

Keep us posted! This is better than Eastenders! (which doesn't say a lot, really... )

seanspotatobusiness on Aug 9 2009, 10:42 PM said:

Well, as of today I have 1 clone out of five which based on digestion looks to be right, I am sequencing it to be sure...So, the only difference in this procedure was the phenol step after PCR, which appears to have been a crucial step in this subcloning. Now I will cross my fingers that the sequence matches and I can be done with cloning for awhile!

hester on Aug 11 2009, 10:32 AM said:

Not sure if anyone is still following but sequence is in... and... Yippee perfect from Start to Stop!!! I believe the PCR cleanup was key!
Thanks for the advice!!!

Glad things worked out. It's why we're here.