Cloning Genes between Vectors by PCR
Be sure that you have enough time so that you can do the entire procedure in one day! Also, try to use
restriction enzymes that are not sensitive to methylation, if possible. Cleavage can be blocked by the
methylation of DNA bases near the restriction site. Consult the restriction enzyme database (REBASE)
or www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_cpg_methylation.asp for
more information.
1. Design primers that flank your gene of interest like so:
5’—GTTTCTT—restriction site—gene—3’
3’—gene—restriction site—TTCTTTG—5’
(use complementary gene sequence here)
Make sure that you have enough overlap with the gene (20-30 bp), since this is the origin of the
primer’s binding affinity. Also be sure that you are in-frame with the tags, both N-terminal and
C-terminal. “Primer3” is a good program to use to design primers. The GTTTCTT overhang gives
the enzymes a supporting substrate around the restriction site.
2. PCR the gene, using 1 min. of extension time per kilobase of your gene and an annealing
temperature 3-5oC lower than the IDT TM of your primers. Cycle the program 20 times. Clean
the PCR product with a QIAGEN PCR Cleanup kit.
3. Perform a double restriction digest on your gene and the vector to which you will insert your
gene. Remember to check ahead of time to be sure that you are not cutting the vector or your
gene in any additional places, and also check NEB website for the best reaction buffer to use
with your restriction enzymes:
www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enz
ymes.asp
To perform digest, mix in a PCR tube:
4 ul 10x reaction buffer
30 ul cleaned PCR product, or 10 ul plasmid (≈ 1 ug DNA)
1.5 ul each restriction enzyme
ddH20 to a final volume of 40 ul
Mix well and incubate for 3 hours at 37oC
4. Clean digest reactions with the cleanup kit. Determine the concentration of your insert and
backbone using the Nanodrop. Perform a ligation reaction (you can use Quick Ligase buffer with
T4 DNA ligase) using 3x excess insert over backbone in a combined DNA volume of 10 ul:
10 ul 2x Quick ligase buffer (mix well by gently shaking before use!)
x ul backbone
x ul insert
1 ul DNA ligase
Mix well and incubate for 15 minutes at room temperature
5. Transform 5 ul into a competent cell line such as TOP10, pick a colony the next day and grow in
LB to generate more plasmid.