IV. Cloning in Plasmid vectors
A. Molecular Cloning Using
TOPO® Cloning
1. Prepare
Plates for transformation:
IPTG (100mM): 0.6g IPTG + 25ml water,
filter-sterilize and store at 4ºC.
X-Gal (40mg/ml): 400mg X-Gal dissolve in 10ml
N,N’-dimethyl-formamide. Cover with aluminum foil and store at -20ºC.
LB
plates with Karnamycin: Add 10g agar, 25g SOC to make 1 liter of medium.
Autoclave 20min 15p. Allow the medium to cool to 50ºC before adding Karnamycin
to a final concentration of 100µg/ml (Stock: 50mg/ml). Pour 20ml of medium into
85mm petri dishes. Let the agar harden. Store at 4ºC for up to 1 month or at
room temperature for up to 1 week. Prewarm plate at 37ºC, add 100µl IPTG (100mM) and 20µl
X-Gal (80mg/ml).
|
Chemical |
Standard Reaction |
|
|
Check |
|
|
Salt solution |
0.5µl |
|
|
|
|
|
TOPO® vector |
0.5µl |
|
|
|
|
|
PCR insert (100ng) |
2µl |
|
|
|
|
|
Total |
3µl |
|
|
|
|
a.
Mix
reaction gently and incubate for 30 minutes (30 seconds to 30 minutes) at room
temperature (22-23°C).
b.
Place
the reaction on ice for 10 minutes and proceed to Transforming One Shot
Competent Cells. Note: You may store the reaction at -20°C overnight.
Start:__________.
a.
Add
1.5µl of the cloning reaction into a vial of half of One Shot Chemically
Competent E. coli and mix gently. Do not mix by pipetting up and down.
b.
Incubate
on ice for 10 minute. Start:__________.
c.
Heat-shock
the cells for 30 seconds at 42ºC without shaking.
d.
Immediately
transfer the tubes to ice.
e.
Add
125µl of room temperature SOC medium.
f.
Cap
the tube tightly and shake the tube horizontally (200 rpm) at 37ºC for 1 hour.
Start:__________.
g.
Prewarm
plate at 37ºC, add 40µl IPTG (100mM) and 40µl X-Gal (40mg/ml) separately, and plate
immediately. Leave the plates at 37ºC for 30 minutes.
h.
Spread
50 µl from each transformation on a prewarmed selective plate and incubate
overnight at 37ºC.
4. Blue/white
screen for recombinants.
i.
Use
teeth pick to transfer white colony to 5ml SOC medium with Karnamycin in Falcon
tube.
c.
Incubate
at 37ºC, 200rpm incubator overnight.
a.
Transfer
700µl bacterial culture into 300µl 50% sterile glycerol in 1.5ml microtube, and
store at -80ºC.
d.
Centrifuge
the Falcon tubes at 1000 g for 5min, decant the supernatant. Follow plasmid
extraction protocol.
Example data sheet:
|
No. |
PCR# |
Gene |
White |
Blue |
Remark |
|
No. |
PCR# |
Gene |
White |
Blue |
Remark |
|
1 |
|
|
|
|
|
|
9 |
|
|
|
|
|
|
2 |
|
|
|
|
|
|
10 |
|
|
|
|
|
|
3 |
|
|
|
|
|
|
11 |
|
|
|
|
|
|
4 |
|
|
|
|
|
|
12 |
|
|
|
|
|
|
5 |
|
|
|
|
|
|
13 |
|
|
|
|
|
|
6 |
|
|
|
|
|
|
14 |
|
|
|
|
|
|
7 |
|
|
|
|
|
|
15 |
|
|
|
|
|
|
8 |
|
|
|
|
|
|
16 |
|
|
|
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