Traditional Cloning
1. Generate insert via PCR with appropriate primers and template
2. Confirm insert with diagnostic gel
3. Purify insert with Qiagen PCR clean-up (elute with 38 µl water).
4. Digest 1hr-O/N with appropriate enzymes in 37o incubator
q.s. 50 µl
Water
q.s. 50 µl
Water
5µl
Appropriate Buffer
5µl
Appropriate Buffer
5µl
10X BSA if required
5µl
10X BSA if required
37µl
Insert DNA
AND
37µl
Vector DNA (~1 µg)
0.5 µl
DpnI
1µl
Enzyme 1
1µl
Enzyme 1
1µl
Enzyme 2
1µl
Enzyme 2
--------------------------------------------After digestion, add 1 µl CIP, inc 37o
0.5-1 hr
5. Gel purify with Qiagen gel purification kit (elute with 50 µl water).
6. Run 10% on gel or use Nanodrop to determine [DNA].
7. Ligate reaction 2 hrs at r.t. or O/N on melting ice in 0.5 ml tube
q.s. 50 µl
Water
5µl
10X ligase buffer
5µl
10X BSA
7.5µl
0.1M dTT
5µl
0.001M ATP
q.s. 100 ng
Vector cut and CIP’d (100 ng)
q.s. 3-4:1 molar ratio to vector Insert DNA (100 ng, 3-4:1 molar ratio)
1µl
T4 ligase
8. Ligate +C cut plasmid
5µl
5µl
7.5µl
5µl
10µl
16.5µl
1µl
10X ligase buffer
10X BSA
0.1M dTT
0.001M ATP
Cut miniprep DNA
Water
T4 Invitrogen ligase 1U (300 cohesive end units)
9. Extract with Phenol/Chloroform/Isoamylalcohol and EtOH ppt:
a. 50µl Ligation reaction
b. 50µl Water
c. 100µl Phenol/Chloroform/Isoamylalcohol
d. Vortex, spin 30s 13K, place 85 µl top layer in new tube.
e. Add 10 µl tRNA (10 µg)
f.
g.
h.
i.
j.
k.
l.
m.
Add 9.5 µl 3M NaOAc, mix
Add 314 µl Ice-cold 100% EtOH
Incubate on ice 15min
Spin 13K 30min
Wash pellet w/ ~500 µl 70% EtOH
Remove all traces of EtOH by aspiration
Dry pellet in SpeedVac ~10min
Resuspend in 6 µl TE
10. Can run the remaining 15 µl of ligation left in extraction on a gel.
11. Chill cuvette, electroporate 3 µl of ligation into appropriate cells and immediately
add 500 µl to 1 ml pre-warmed 2YT. (1.4 kV, 200 , 25 µF)
12. Incubate 37o C 1hr
13. Streak 50 µl on one plate and spread remaining at 100 µl/plate on multiple plates
with appropriate selective agent.
14. Allow all plates dry by leaving lids slightly ajar before incubating overnight.
15. Pick individual colonies for patching and miniprep
16. Digest to check for insert and send positives out for sequencing.
17. Store at -80o C stocks of confirmed clones.
Notes:
Electroporator settings: 1.4 kV, 200 , 25 µF
10X Ligase buffer
30µl
1M Tris pH 7.6
7.5µl
1M MgCl2
112.5µl
Water
1M dTT
154.25 mg
dTT
1 ml
water
For gel purification, use thick tooth side of comb.
Stock Ethidum Bromide is 10 mg/ml.
Make 900 ml 1X TAE + ~20 λ EthBr for large gel running apparatus.
Load 5 λ of 100 γ NEB 1 kb ladder
0.8% agarose
EthBromide
small gel (70 ml TAE)
0.56 g
2.8 λ
large gel (150 ml TAE)
1.2 g
6λ