Cloning after PCR
Insert:
1, PCR amplify a desired fragment from either miniprep DNA, genomic DNA, cDNA from mRNA, or gene synthesis
2. Run gel to isolate desired fragment
3, Cut out band
4, Add 500~800 ul QG Buffer
5, Dissolve gel at 50~60 oC
7. Purify using a mini spin column
8, elute with 25 ul H2O
9, Add 3 ul of 10X Buffer (see NEB chart for NEB buffer or use FastDigest buffer)
10, Add 0.5~ 1 ul restriction enzyme
11. Digest at 37 oC for 30 min to 60 min.
Vector
From step 9, digest ~150 ng of vector with the same enzymes or compatible enzymes for 30 min at 37 oC.
12. run gel cut out desired vector band
13, Add QG, dissolve
14, Add digested insert, from step 11 mix.
15, Purify with a spin column
16, Elute with 25 ul H2O
Ligation
Add 3 ul 10 X T4 ligase buffer, 1 ul T4 DNA ligase
Ligate at RT for 30 min
Transformation