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Digestion and Ligation with Restriction Enzymes
Author: Alex Kuo, updated date: , view: 4, Q&A: 0
Tags: Molecular Cloning, Ligation and Restriction Enzyme

Procedure

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


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