Direct ligation with oligo's
For cloning fragments up to 120bp into a restriction site without PCR or restriction of the fragment’s ends.
Design complementary forward and reverse primers. Oligo’s should be identical apart from the primer ends that should be designed as restricted sites. (Examine the required restriction sites in Rebase )
For example: if we choose to clone the fragment into a BglII site
A^G A T C T
T C T A G^A
we need to add sticky ends on both ends of the oligo:
Forward oligo will read:
Reverse oligo will read:
1. Resuspend oligo's at 0.5 ugr/ul in H2O
2. Take 1ugr of each oligo into an eppendorf tube
3. Add water to final volume of 5ul.
4. Heat 5min 90ºC
5. Spin down and leave at RT for 5 min
6. Leave 5 min on ice.
7. Ligate 1ugr of insert with 100ngr of vector (in 10 or 20ul)
Do not de-phosphorylate the vector!!!!
The insert is de-phosphorylated, hence no problem with too much insert in reaction!