Download Ligation Independent Cloning

Ligation Independent Cloning
Written By Mariana Lopez
Last Edited by Andy McMillan Jan. 15, 2015
Summary based on Eschenfeldt et al. “A Family of LIC Vectors for High-Throughput
Cloning and Purification of Proteins” Sharon A. Doyle (ed.), Methods in Molecular Biology:
High Throughput Protein Expression and Purification, vol. 498
Step 1: Primer design for PCR amplification of genes
Example for using pSGC23-His and related plasmids. Other plasmids may need different primer
sequences
Sense Primer: 5’-TACTTCCAATCCAATGCCxxxxxxxxxxxxxxx
Antisense Primer: 5’-TTATCCACTTCCAATGTTAyyyyyyyyyyy
xxxxx – refers to a series of nucleotides from the target gene
yyyyy - complementary of the gene sequence being amplified from its 3’ end
PCR mix (50 L)
HiFi Buffer (5x)
Phusion Taq polymerase
dNTPs (20 M)
Template DNA
Forward primer
Reverse primer
dH2O
10L
1 L
2 L
1 L
1 L
1 L
34 L
PCR conditions for Phusion Taq polymerase
95 C
------------95 C
55 C
72 C
------------72 C
4 C
3 min
30 sec
45 sec
90 sec
35x
10 min
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Step 2. PCR purification (Qiagen or Epoch Kit), and follow protocols supplied with the kit.
Step 3. Determine DNA concentration of your sample(s).
Step 4. Treat your sample(s) with T4 DNA polymerase
To a 0.4 Eppendorf tube on ice add:
PCR product 600 ng
10x NEB Buffer 2
dCTP (100 mM)
DTT (100 mM)
ddH2O
T4 DNA polymerase
x L (x+y=32L)
4 L
1 L
2 L
y L (x+y=32L)
1 L
Incubate reaction mix at room temp for 30 min
Inactivate T4 DNA polymerase by heating reaction mix at 75C
Step 5. Prepare the vector for Ligation Independent Cloning (LIC)
Digest vector with SspI (or other restriction enzyme depending on plasmid)
To a 1.5 Eppendorf tube on ice add:
Vector DNA (1 g)
10x NEB Buffer 2
SspI restriction enzyme
100x BSA
ddH2O
x L (x+y=51.4 L)
6 L
2 L
0.6 L
y L (x+y=51.4 L)
Incubate reaction mix at 37C for 2 hours.
Run gel to confirm complete digest. Purify using PCR purification kit.
Step 6. Treat your SspI digested vectors with T4 DNA polymerase
Vector (200ng) (Assume 25% loss)
10x NEB Buffer 2
dGTP (100 mM)
DTT (100 mM)
ddH2O
T4 DNA polymerase
x L (x+y=32 L)
4 L
1 L
2 L
y L (x+y=32L)
1 L
Step 7. LIC Annealing
15ng Vector DNA
45ng PCR product
Mix gently by tapping and incubate on ice/30 minutes
Transform 5-10ul of annealed products in CaCl DH5a cells.
Step 8. Colony PCR to determine ligation success
Pick individual colonies and resuspend in 30ul of ddH2O. Use 3ul of resuspended cultures in
PCR reaction using gene specific primers (from step 1)
PCR mix (20 L)
Thermopol Buffer (10x)
Taq polymerase
dNTPs (20 M)
Resuspended colonies
Forward primer (10uM)
Reverse primer (10uM)
dH2O
2 L
0.25 L
1 L
3 L
1 L
1 L
12 L
PCR conditions for Taq polymerase
95 C
------------95 C
55 C
68 C
------------68 C
4 C
5 min
30 sec
45 sec
90 sec
28x
5 min
