Next time I would try sub-cloning the DNA fragment into a TOPO vector for easier cloning.
This is much easier, about 50X easier (sticky end cloning vs blunt end cloning)!
If you already have primers for your DNA fragment, look into the TOPO cloning or TA subcloning vectors from Invitrogen.
My protoocol for Blunt end cloning is to use Klenow to blunt the end of the PCR product and clone into a site that I have a Blue:white screen for inserts in your favourite vector.
Some tricks for Blunt end cloning:
The most important thing about blunt end ligations is the concentration of ends and the number of units of Ligase added.
I found that sticky end ligations only require a fraction
of the ligase that blunt end DNA fragments need so that DNA Ligation go es efficiently.
So I put in 5 Units of ligase for blunts. Higher if I can get it. I once got some from BM that was 8 units/ ul. Now they seem to be standardizing on 5U/ul. BLUNT END CLONING PROTOCOL
- I use the following method to purify my PCR product from a gel (however the problem is the yield after is quite low) using Girvitz et al (1980) Anal.Biochem. 106:492 as described in Maniatis, Fritsch and Sambrook (1982) pg 168.
- I then do the klenow and ligation reaction in a single tube. I use 200ng of vector for blunt ends, 1 x Nick Translation buffer made with MgCl2 not Mg(So4)2, about 3X M excess purified PCR fragment if possible, 5 units of klenow in 20 uL total volume.
- Incubate reaction for 5 min at room temperature (RT) and then add dNTPs to 100uM
- Incubate for 10 more minutes.
- Then add 1/2 ul of 50 mM ATP not dATP.
- Add 1/2 ul of 250 mM DTT
- 1 uL of 5-8 units (U) of T4 DNA ligase (Use Highly Concentrated Ligase!)
- Make up to 25 uL with purified H20 (even better dont add much water - decrease total volume, keep this concentrated for blunt end)
- Ligase at 12 degrees C overnight ( do not use a heating H20 bath located in the cold room as this causes condensation problems and prematurely stops the ligation. I use a ligation refrigerator or a cooling water bath)
- Next day (morning), Add 2.5 uL 3.0M Na acetate pH 6.0 and 60 ul of Cold Ethanol PTTE and dissolve in TE and Electroporate.
- I do 20 minis and if I get no inserts I start again!
Be sure that your PCR product is kinased or your primers have been to start with. I also SAP antarctic phosphatase is better though, my vector well to minimize the self ligation.
If when you transform your bacteria you do not get any clones the first time it is probably due to low concentration of ends of your PCR product.
200 ng of vector in 20 ul of ligation volume is correct for vectors at the size of pUC or even pBR322 for blunt ends.
If you dont have alot of PCR product you can always reduce the ligation volume and scale everything else down accordingly including the vector DNA. I have done ligations in 5 ul that have worked!
If you SAP your vector and put in about a 3X molar excess of ends of fragment and 5-8 units of ligase I find it works everytime.
I don't want to give you the impression that I calculate the molar quantity of ends for each fragment I am trying to clone. The 3x is not always necessary, it seems that smaller fragments are easier to blunt end clone than larger ones so the larger the fragment the closer attention I pay to this. In your case with a 3 kb fragment I would probably try and get a hold of 600 ngs of purified insert to clone with 200 ngs of vector.
If you can only get 400 or even 200 scale down the ligation volume.
One last thing I always try and purify my PCR products from a gel as this seems to decrease the probability of getting UCOs ( unidentified
cloning objects). These are small fragments that dont usually relate to the PCR product itself, but by the nature of the process are found in
small quantities in the reaction ( the smear region of the gel) At least if you can size purify you can cut down on UCOs.