Biology Reference
In-Depth Information
5.1 Direct
Bacteria PCR
Direct bacteria PCR is a technique which allows selecting clones
from transformed bacteria cells that contain the desired DNA
without prior plasmid preparation and enzymatic tests.
1. Pick at least 10 individual colonies. Pick also 2 colonies form
the control plate.
2. Grow colonies for at least 4 h (37 °C, 190-220 rpm) in 400
μ
L
LB-medium containing 100
μ
g/mL ampicillin/carbenicillin
in a 1.5 mL reaction tube.
3. For PCR mix the following components on ice together; always
adding enzyme last. For multiple samples, make a master mix
and aliquot 10
μ
L in each well of a PCR-plate (also on ice):
10× RDA buffer
1.5
μ
L
MgCl
2
1.2
μ
L
dNTPs (10 mM each)
0.3
μ
L
Primer sense (10
μ
M) (pLKO-forward/pLJM1-forward)
0.7
μ
L
μ
μ
Primer antisense (10
M) (pLKO-reverse)
0.7
L
H
2
O
4.6
μ
L
Taq
1
μ
L
4. Add 5
L of the bacteria suspension. Don't forget controls
(H
2
O instead of bacteria suspension).
5. Cover wells with mineral oil.
6. Use the following PCR conditions:
μ
95 °C
3 min
95 °C
30 s
53 °C
30 s
30x
72 °C
30-45 s
72 °C
5 min
μ
7. To check the outcome, apply 5
L of each PCR reaction to a
1.5 % agarose gel.
5.2 Preparation
of Plasmid DNA
1. Set up overnight cultures of positive candidate colonies.
2. Purify the miRNA construct using an endotoxin-free plasmid
purifi cation kit (
see
Note 14
).
3. Confi rm the insert by sequence analysis of the miRNA
construct using the direct bacteria PCR primers (
see
Note 15
).
