DIGEST & LIGATION W/RESTRICTION
ENZYMES
Vector: pENTR 3C
Look up REs on NEB to see which buffers
are compatible
i.e. NotI and EcoRI Buffer 3.1
For one enzyme (20 ul):
10 ul vector
2 ul enzyme
2 ul buffer
6 ul water
Make sure enzyme is 10% of final
volume. Buffer is at 10x in -20 “Cloning Reagents” box.
(For 2 enzymes, final volume will be 40
ul)
2 hours @ 37o and keep at
-20o
Purify PCR product with kit and purify
plasmid with gel.
10x ligase buffer (T4)
T4 ligase in blue box = 1 ul per
ligation
Use 3x more insert by molar ratio.
2 ul vector
6 ul insert
1 ul buffer
1 ul ligase
RT overnight
Transform into 100 ul DH5alpha
Ice 30 min
37 2 min
Ice 2 min
Add 900 ul SOC media
Shake @ 37o 1 hour
Spin down and plate
RECOMBINATION
WITH LENTIVIRAL PLASMID
1 pENTR
1 destination vector
6 TE buffer
2 LR clonase
10 ul reaction
25o 1 hour
Add 1 ul proteinase K
37o 10 minute
Transform into
STBL3
pLenti puro Amp