Accidentally using 2 sites w/ compatible ends - Ways to cut down on background? (Apr/19/2011 )
Hi everyone, so I'm trying to clone some genes for the first time, and in my genius I decided to cut my vector and insert w/ BglII and BamHI, which give compatible sticky ends. I'm trying to clone in a PCR product with the restriction sites added onto the primers. We have one vector that we would like to clone a lot of genes into, so I wanted to digest about 5ug of it to have plenty for ligations. My steps were this:
For the insert, I did PCR, did a RE digest for 4 hours, then ran on a gel and had one pretty band at the right size I cut out and did a gel purification kit on.
For the vector:
Set up 5 different identical double digests, each with 1ug of plasmid, each 50 ul total, and let digest for 4 hours at 37.
Since BamHI-HF said it may hold on to the DNA tightly, I combined these 5 digests and ran through a Qiagen PCR Purification Kit in order to get the REs out of the sample.
I then split the eluate from this, 50 ul, into two tubes for CIP. I had supposedly 2.5ug DNA in each, I used 2.5 units of CIP in each, and had a total volume of 50 ul in each tube. I let this go for 40 minutes at 37, then ran this on a gel and saw bright bands at my expected size. I cut these out, used a qiagen gel purification kit, then ran 1 ul of both the vector and the insert on a gel to check the concentrations. I set up the ligation at a 1:4 molar ratio vector:insert, and also had a control with no insert. This morning I have just as many colonies on my control as on my ligation plate.
Is perhaps my CIP treatment not thorough enough and I need to redo it for longer, or is something else happening? Any ideas on this would be very appreciated.
-Jesse
i would suggest doing all the essential controls to be able to evaluate what is actually going on!
This means:
1) Vector+Insert+Ligase
2) Vector+Ligase (to quantify undigest+re-ligated vector)
3) "Digested" Vector only (quantifies undigested vector)
4) Insert only+Ligase (put that on a gel, this should give a ladder with discrete bands at the size of various multimers)
All of the above should give you a feeling for what is going on and where your background originates from. CIP treatment is always tricky since it is hard to inactivate. If you have a carry over from the CIPed vector to your reaction this will mess up your whole ligation. I personnaly perfere using shrimp alkaline phosphatase or antarctic phosphatase since these enzymes can be heat inactivated.
Hope this helps!
Regards,
p
JesseC on Tue Apr 19 15:05:56 2011 said:
Hi everyone, so I'm trying to clone some genes for the first time, and in my genius I decided to cut my vector and insert w/ BglII and BamHI, which give compatible sticky ends. I'm trying to clone in a PCR product with the restriction sites added onto the primers. We have one vector that we would like to clone a lot of genes into, so I wanted to digest about 5ug of it to have plenty for ligations. My steps were this:
For the insert, I did PCR, did a RE digest for 4 hours, then ran on a gel and had one pretty band at the right size I cut out and did a gel purification kit on.
For the vector:
Set up 5 different identical double digests, each with 1ug of plasmid, each 50 ul total, and let digest for 4 hours at 37.
Since BamHI-HF said it may hold on to the DNA tightly, I combined these 5 digests and ran through a Qiagen PCR Purification Kit in order to get the REs out of the sample.
I then split the eluate from this, 50 ul, into two tubes for CIP. I had supposedly 2.5ug DNA in each, I used 2.5 units of CIP in each, and had a total volume of 50 ul in each tube. I let this go for 40 minutes at 37, then ran this on a gel and saw bright bands at my expected size. I cut these out, used a qiagen gel purification kit, then ran 1 ul of both the vector and the insert on a gel to check the concentrations. I set up the ligation at a 1:4 molar ratio vector:insert, and also had a control with no insert. This morning I have just as many colonies on my control as on my ligation plate.
Is perhaps my CIP treatment not thorough enough and I need to redo it for longer, or is something else happening? Any ideas on this would be very appreciated.
-Jesse
How long can you keep your digested vector for? I am never confident with using older digested vectors. I prefer to do it more often, as I need it, than to have a stock. Maybe your vector wasn’t digested properly. That is a lot of DNA and if enzymes are used by other people you never know where they have been.
Anyway, doesn’t matter how often you digest that vector you will always face same problem. Maybe if you take pDNA’s advice and use different alkaline phosphatise to treat your vector but instead of insert ligate compatible primers composed of unique restriction enzyme sites (these primers will have to have phosphates added to them and be designed as if they were digested with BamHI and BglII, or any of the two, meaning single digestion). That way you can design new MCS and eventually use enzymes which do not produce compatible ends.
Might be a bit lengthy but saves you a lot of trouble and time down the track. Trust me. 