|
Home :: Technical Resources
|
Technical Resources
GeneReleaser® Tips
|
We have found that certain actions are essential for
The proper performance of GeneReleaser®. These are
described below.
- The sequence of reagents additions of the reaction
Components is critical. Additions should be performed
in the following order:
- Use 1µ of whole blood or cells at a density of
~108/ml.
- Add 20µ of GeneReleaser® (for standard 100µ
amplifications).
- DO NOT vortex or mix components after steps
A&B above!
- Lyse the cells using the thermocycle program
protocol.
- Add amplification reagents (DO NOT vortex or mix).
- Perform amplification. NOTE: It is very important
that the very first denaturing step of the first cycle
Be at 94°C for 2-5 minutes depending on brand of
cycler, reaction volumes, etc.
- The specimen and GeneReleaser® volumes may
be adjusted. However, no less than 5µl of GeneReleaser®
or more than 5µ of specimen should ever be used.
- The volume of GeneReleaser® used to accomplish cell
lysis should be compensated for by deducting an
equivalent volume of H2O from the components of the
amplification reagents in order to maintain their
appropriate concentrations in the final reaction volume.
- Amplification reaction volumes may be reduced from
the typical 100µ volume to as little as 25µ as long as
proportionate reductions are made with respect to
specimen and GeneReleaser® volumes.
- The original thermocycle program has been modified
to an 80°C hold in order to obtain better amplification
from denser tissue materials and to facilitate an initiation
of the amplification cycles under conditions which
minimize non-specific annealing of primers.
- It must be emphasized that if, upon use of this product,
the expected bands are not observed, then a magnesium
titration should be performed. If this fails to produce the
desired bands, then reduction of the annealing temperature
by 5-10°C should be employed in conjunction with a
magnesium titration. If either of these should fail, we
will be glad to develop an optimized procedure with you. in developing an optimized protocol.
- If the GeneReleaser® treated specimens cannot be amplified
after performing the procedure, specimens may be stored at either
4°C or -20°C until they can be amplified. Prior to amplification
stored specimens should be heated to 80°C and the amplification
begun using steps 10-12 of the GeneReleaser® protocol.
|
|
Microwave Protocol |
Note: The 0.2ml tube racks provided by
Perkin-Elmer for use with their 9600 and 2400
instruments are NOT microwave compatible.
THEY WILL MELT!!
- We have found that microwave treatment of
Specimens affords a more rapid sample
preparation and facilitates the amplification of
the more intractable types of specimens.
- Place 1µl of specimen with 20µl of
GeneReleaser® into either a 0.5ml or 0.2ml
PCR tube. NOTE: It is extremely
Important not to use tubes any larger than
these; as the samples will be boiled away or
larger tubes will rupture!!
- Unlike the thermocycle program, vortex the
tubes containing specimen and GeneReleaser®
for ~10-30 seconds.
- An oil overlay is optional.
- Placed closed tubes in polyethylene or
polypropylene racks.
- Place rack in microwave oven and heat at
Maximum power setting for 5-7 minutes. 5 minutes if wattage is 900 or higher, 7 minutes
if wattage is 500. 4500 watt-minutes is the
optimum.
- Remove rack from microwave and place tubes
on preheated thermocycler at 80-90°C.
- Add PCR master mix and begin amplification
cycles. NOTE: It is very important that the
very first denaturing step of the first cycle
be at 94°C for 2-5 minutes depending on
brand of cycler, reaction volumes, etc.
If you ever have any questions regarding the use
of GeneReleaser® or reaction conditions please call
or fax us. If you can spare either specimens
(noninfectious) or primers, we will gladly assist
in developing an optimized protocol.
|
|
|
