DNA Ligation Problem Double Digestion

[Denise]

Hi,
I have a new problem I didnt get colonies after transformation.



I cloned a 1.5kb DNA piece into the vector/plasmid MCS pcDNA3.1+ however, but when I tried to put another 1.5kb piece into the MCS I cant get any colonies.

I am conducting PCR on the piece, then I double digest the PCR product and the plasmid by double digestion with BamHI and HindIII overnite at 16C.

I then conduct ligation at room temperature with the T4 DNA ligase overnight.

I am ligating at a ratio of DNA to Plasmid at about 3:1.

And the total amount of insert use for transformation is 5ul/50ng DNA.

What could I be doing wrong?
thanks

[Qiang]

Hi Denise,

Basically it is NOT recommended to perform a double digestion in case of BamH1 and Hind3 due to the strong "Star activity" of BamH1 in NEBuffer 1,2, or 4.

If you used NEBuffer systems, and did double digestion in NEBuffer2+BSA, this is then NOT supprise for your result.

I suggest to digest sequentially: in NEBuffer2 digest first with Hind3 for 1-2hrs>>add NeBuffer3, BSA and digest with BamH1 for 1-2hrs. >>(optional: gel purification)>>(optional: dephosphatase treatment on vector)>>then perform ligation with NEB T4 ligase (optional: vector + H2O as control) for 10 min>>transformation. This should work!

Best and good luck!

Qiang

[jiajia1987]

I am sorry to add onto this but I would like to clarify something.

BamH1 has STAR activity so it is not recommended to do an overnight digestion regardless of which NE Buffer is used, right? I tried out an overnight digestion using BamH1 and Nde1 and I got a huge smear on the gel the next day.

[jiajia1987]

I am sorry to add onto this but I would like to clarify something.

BamH1 has STAR activity so it is not recommended to do an overnight digestion regardless of which NE Buffer is used, right? I tried out an overnight digestion using BamH1 and Nde1 and I got a huge smear on the gel the next day.

[trypsin]

Did you purify the pcr-droduct after the pcr?
Somebody told me that some polymerases, e.g Phu have a stronger exonuklease activity than other so that they cut your sticky ends after digestion.
the Buffer for the digestion also could be a problem. the instructions of the enzymes from NEB have an information about the enzyme activity depending on the Buffer.
when you use to different enzymes you need to check if these have good activities (75-100%) in the same buffer.

bye, trypsin

[arunab129]

dear all,

can any one help me in my problem. i am working with pET DUET SUMO vector to clone my gene. and my restriction sites are BamH1 and Kpn1.i cant able to get the clone. i have a doubt whether double digestion of vector is occuring with the two restriction enzymes or not. can any one please tell me how can i perform double digestion.

[SebaQ]

Originally Posted by arunab129

dear all,

can any one help me in my problem. i am working with pET DUET SUMO vector to clone my gene. and my restriction sites are BamH1 and Kpn1.i cant able to get the clone. i have a doubt whether double digestion of vector is occuring with the two restriction enzymes or not. can any one please tell me how can i perform double digestion.

The most important thing you have to check are the enzymes activity conditions. Not all the digestion enzymes can work on the same buffer, or at the same temperature.

Some New England Biolabs digestion enzymes (the ones I usually use) can be used together within the same temperature, time and buffer, so you can simply add both enzymes to the NEB Buffer, add the BSA, DNA and then incubate the reaction and have a double digest in only one reaction.

If you want to double digest with two enzymes that don't act at the same conditions, or have star activity or something that makes your one-step double digestion troublesome, you will have to digest with one enzyme first, then purify the DNA and digest with another enzyme, then re-purify to ligate with your vector to transform.

But, are you completely sure the double digestion step is the one compromising your cloning? There are many other steps than can affect your cloning, like the ligation step, the transformation step, the efficiency of your bacteria, etcétera.

Best regards!

[Nimafakour]

Hi every body, I tried to insert a fragment in to plasmid pCW101 which is cut by hindIII and salI. the fragment was cut with similar Restriction enzymes. I cloned the plasmid in E. coli. But today, when I extract the plasmid and digest it again. I couldnt detect my fragment on the gel. what could be the reason.

[atin]

Originally Posted by jiajia1987

I am sorry to add onto this but I would like to clarify something.

BamH1 has STAR activity so it is not recommended to do an overnight digestion regardless of which NE Buffer is used, right? I tried out an overnight digestion using BamH1 and Nde1 and I got a huge smear on the gel the next day.

I am facing a completely different problem!!! I digest my vector pQE 30 with Bam HI for about 4 hours and still get uncut plasmid !! I had to go for an 8 hour digest (although not recommended) to get it completely digested. What is the most surprising thing in this? I used about 6 units of enzyme and my plasmid was only 1ug!!!
Why is it said that BamHI gives instantaneous digestion??? What if I incubate for long (as I have done)???????????