Cloning problems - (Jun/29/2010 )

Hello,

I have been in cloning hell for a few months now and just wanted to ask for some help. I have been trying to clone a 3000bp insert into about a 3800bp pCMV-HA vector. I cloned the insert into pcR2.1 TOPO vector. I initially intended to use Bgl11 and Not1 to cut but my TOPO vector has another Bgl11 site within it that affects my gel purification of the insert as they end at almost the same size and have tried using lower conc gels but no success. So the digestion seems to be going fine for both but the problem is that the ligation is just not working well. I get more clones on my background than my vector+insert.Which most probably means my vector is religating or I am having insufficient digestion. Also because of the Bgl11 problem with the TOPO vector, I have had to cut the TOPO vector with BamH1 and Not1 and then ligate into Bgl11 and Not1 sites in the vector because my PI tells me that even if I cut my insert with BamH1, it will still be able to ligate to the Bgl11 site.

This is pretty much the summary and if anyone has any suggestions that would be really helpful.

Hi,
I am not sure if I understand correctly your story but what I can say is that is you are cutting the vector with two different enzymes there should be no relegation. Also are you sure that you can cut the insert with BamH1 and then ligate to the Bgl11 site? Sound weird to me!
Are you using CIP? When you have different restriction site in not useful and can damage your vector end.
I hope this is helpful...

milla on Jun 29 2010, 08:32 AM said:

Sorry if it is not as clear. Well Bgl11 and BamH1 actually have compatible ends so you can do ligation with those 2 ends
No, I did not use any CIP. I was actually considering using it since it could be that my vector was religating

From one denizen to another, 'Welcome to Cloning Hell!' . You stay, no matter how long, always feels like an eternity. It will be a trilling and amazing experience, spanning the depths of blackest despair to the euphoric heights of false victories. Please leave an offering of a kit-kat to the Goddess of Molecular Biology before you leave.

Now with initial introduction done, lets what can be done to expedient your departure from Cloning Hell. If I understand things correctly, the problems are as follows.

Insert is in TOPO vector.
Insert is being cut out using BglII and NotI
Insert vector has second BglII site giving a fragment which is similar in size to desired insert.

There are a few things you can do
1- use 3 restriction enzymes in the digest. The 3rd enzyme is used to cut the unwanted fragment to a smaller size.

2. - PCR amplify your desired insert, (primers are cheap now). You can then add your desired restriction sites to the primers.

Lots of background from vector religation.
-this is rather odd given that the vector has been cut by two restriction enzymes.
-this might hint that the double digest isn't going too well.

What is the distance between the BamHI and NotI site. Please note that RE require a few basepair skirting their restriction site before the enzyme will cut the DNA efficiently. NotI requires a minimum of 8b. BamHI about 2bp.

How long are you cutting the vector? Could you also write down the digestion formula used.

Lastly, if you are going to dephosphorylate, please note that CIP under conditions of overdephosphoryaltion has a nasty tendency to render vector molecules unligatable. So do not overdephosphorylate with CIP. Antarctic phosphatase is more forgiving.

Second most of what Perneseblue says. Especially note the overhang length required for NotI digestion. You can dramatically reduce or eliminate background vector by using PCR for your vector, rather than your insert. PCR the vector with whatever restriction enzyme sites you need in the primers (remember to add sufficient 5' overhang). PCR cleanup, then digest with a mixture of your two REs for the ends, along with DpnI, to cut the template DNA used for your PCR reaction. This will give you linear DNA with the correct ends, and essentially zero background. Cut your insert with compatible enzymes, ligate, go. This is especially effective if your final vector has a different antibiotic resistance than the vector containing your insert, since you select against the insert only vector as well.

First I want to say that the BamHI BglII trick works, I have done it myself.

If you really want to keep the BglII site, what you can do is digest your TOPO vector with NotI and another enzyme that is outside the BglII site, gel purify that fragment, digest with BglII then gel purify again.

For your vector, I just want to make it clear that vectors with two different ends CAN re-ligate by more than one vector ligating together. I suggest you CIP your vector (I usually CIP for the last hour of my digestion) which will reduce a lot of background.

I would also check if both your enzymes are cutting like other's have suggested. Do some control digestions of your vector:

1 with only BglII
1 with only NotI
1 with Both BglII and NotI

however, since your fragment from the TOPO vector is falling out after digestion, it doesn't seem like your problem is undigested vector. The only reason I can think of would be that mentioned above about the base pairs between restriction sites.

Thank you all for the suggestions. THis has been hell and is still hell for some time...Well, to answer your questions. I do not think it is the number of bases, because I actually PCR added on the restriction sites to the insert before inserting into the TOPO vector. I made sure that both sites had sufficient overhang especially Not1, which is notorious for this.

I will take into consideration cutting with another restriction enzyme within my unwanted Bgl11 restriction fragment within the vector.

I have actually managed to see less background on the vector in my last try. However when I digest the supposed product with EcoRI, I am getting a weird gel pattern. I sent in the stuff for sequencing and they could not really get any product so I am trying to troubleshot that to make sure that it is not the primer or the template. Arghh! Will see in a day or so. I resubmitted the stuff.

As to my digestion formula:

For the pCMV-HA vector, which I am adding my insert to:
5ug of vector
2.5uL of Not1
Buffer
Water to 50 uL and then I incubate this for 2 hours

After the 2 hour incubation, I add in 2.5uL of Bgl11 and incubate for another 2hours

Initially, I added all the REs together but because of the religating, I thought that this might be a better protocol.

Thank you all for your help.

ropa on Jul 3 2010, 06:34 PM said:

Hmmm....you are using too much enzyme. Restriction enzymes come in glycerol as a preservative. However glycerol also inhibits enzyme activity and cause some enzymes to exhibit star activity (reduced restriction site specificity). As a rule of the thumb, total concentration of restriction enzyme should not exceed 5%.

Reduce the volume of enzyme used (1uL should be enough) or increase the volume of the restriction digest.

How far apart are the NotI and BglII sites on the pCMV-HA vector?