Digestion and Ligation with Restriction Enzymes

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 @ 37^o and keep at -20^o

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 @ 37^o 1 hour

Spin down and plate

RECOMBINATION WITH LENTIVIRAL PLASMID

1 pENTR

1 destination vector

6 TE buffer

2 LR clonase

10 ul reaction

25^o 1 hour

Add 1 ul proteinase K

37^o 10 minute

Transform into STBL3

pLenti puro Amp