Reagents
Provided by Mycoplasma Plus PCR Primer Set (Stratagene, #302008, $299):
- Rehydration buffer, store at RT
- PCR primers, rehydrate with 200 µl rehydration buffer, store at -20°C
- Internal control, rehydrate with 100 µl rehydration buffer, store at -20°C
- Positive control, rehydrate with 100 µl rehydration buffer, store at -20°C
- StrataClean Resin, store at 4°C
To be supplied by user:
- 0.6 ml tubes or 1.5 ml screw cap tubes
- PCR reagents (i.e. dNTP, Biolase Taq, MgCL2,10x reaction buffer)
- UV-irradiated ddH2O
- 2% agarose gel (3 g agarose, 3ml 50x TAE, 147 ml H2O, 75 µl (0.5 µg/µl) Ethidium Bromide)
- loading buffer
Protocol
A)
Template Preparation
- Centrifuge all supernatant samples to pellet any cell debris as this can
inhibit the PCR.
- Transfer 100 µl cell supernatant to a 0.6 ml tube (screw cap tubes for viral
supernatants). Close tube tightly to prevent opening during heating step.
- Boil (or heat to 95°C) supernatant
for 5 minutes. Spin tube briefly (2-5 seconds) in a
microcentrifuge.
- Resuspend the StrataClean Resin by vortexing for
30 seconds until no pellet remains. Add 10 µl
of resin to the extract. Mix the resin and the
extract by gently flicking the tube. Briefly
centrifuge for 5-10 seconds to pellet the resin.
- Remove the supernatant to a fresh tube and dilute (optional)
1:10 with UV-irradiated water Per PCR, 5 µl
of diluted template is required. Avoid aspirating the
resin into the aliquot. Store the template at 4°C until
use.
B)
Mycoplasma PCR
The
amount for one reaction for Mycoplasma PCR is 50 µl.
Prepare a premix of reagents for multiple reactions.
The amount for the premix reagents is 45 µl
per reaction. The amount of each reagent for
premix is as follows:
Water
32.1 µl
10x RB
5.0 µl
MgCl2
(25 mM) 3.0 µl
DNTP
(20 mM)
0.5 µl
Primers
(25 µM)
2.0 µl
Internal
control
2.0 µl
Biolase
(5 U/µl)
0.4 µl
45
µl/reaction
- Calculate volume of premix needed for all samples, one positive control,
one negative control (water) and one extra reaction.
- Thaw PCR reagents from freezer (except Biolase Taq). Make premix in one tube.
- Add Biolase Taq into premix tube, mix and aliquote into
PCR tubes.
- Add 5 µl test sample, positive control or
negative control into the tubes.
- If not using thermal cycler with heated lid, add
2 drops of mineral oil.
- Keep tubes on ice until ready for cycling.
- Start PCR cycling. The following PCR program yields
optimal amplification of the 874bp product:
Segment
|
Cycles
|
Temperature
|
Duration
|
|
1
|
1
|
94°C
|
2 minutes
|
|
|
|
50°C
|
2 minutes
|
| |
|
72°C
|
2 minutes
|
|
2
|
40
|
94°C
|
1 minute
|
|
|
|
50°C
|
1 minutes
|
|
|
|
72°C
|
2 minutes
|
- Store samples at 4°C until ready to run agarose gel.
- Make 150 ml of 2% agarose gel.
- Prepare samples for loading: 2 µl 5x loading buffer,
3 µl H2O, 5 µl PCR products (or 2 µl 1kb,
φx174 or HindIII marker).
- Load the samples and run the gel for 1 - 1.5 hours at 100 volts.
- Visualize banding pattern: positive control = 874bp,
internal control = 1000bp.
For specifics on appropriate handling and waste procedures please see the
online chemical SOPs or our waste and spill notebook located in
room 352.
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