How to prepare your DNA for fly injections
Good quality DNA is a prerequisite for good
injections. CsCl preparation is the preferable method for fly transgenic
work. However, for DNA construct less than 10 kb, Qiagen prep in most
cases is good enough. We found it's a lot easier to produce enough
quality DNA using 6 Miniprep columns rather than one Midi- or Maxi- prep
using Qiagen plasmid purification kit.
For P-element construct and helper DNA construct, please prepare 20
microgram DNA in 20 microliter water (final concentration is 1 microgram
per microliter) for each of them.
Please follow the FlyInjection Laboratories recommended quality
assurance protocol listed below (FILTRED Test) to do a quick DNA quality
test before you send us your DNA. If you send the DNAs by Fedex
(preferable shipping method) overnight delivery, you don't need dry ice.
FlyInjection Laboratories Thirty minutes Restriction Enzyme
Digestion Test (FILTRED Test) for DNA quality
The DNA quality is quite important in fly injections. It's always a good
practice to check your DNA prep by gel before you send them out. At
FlyInjection Laboratories, we developed a quick FILTRED Test for
checking the quality of your DNA. The protocol is as follows.
1. Select one restrictions enzyme that will cut the DNA construct at two
or three sites so the complete digestion will produce two or three clear
distinguishable bands.
2. Dilute the selected restriction enzyme in 1 X reaction buffer to a
final concentration at 0.5 unit per microliter (0.5u/ul).
3. Set up the following reaction mix:
0.3 microgram DNA construct
2 microliter 10 X reaction buffer
Add H2O to final volume of 18 microliter
Add 2 microliter (1 unit) of the diluted restriction enzyme prepared in
step 2. Mix the reaction.
4. incubate at 37 degree celsius (or other appropriate temperature for
certain restriction enzymes) for thirty minutes and then run the sample
on agarose gel.
You should get clear sharp linearized bands. If you get fuzzy band or
extra smaller band, please reprep your DNA. However, a little amount of
genomic DNA contamination in the prep (usually they won't run far and
are close to the gel wells during gel electropherisis), by our
experiences, doesn't interfere with fly injections.
This test is very sensitive. If your DNA sample contains more salt,
protein, or not disolves well, you will not get clean cut for 30 minutes
digestion, and should re-prepare your DNA. Please call or email us if
you have more questions about DNA prep.
Contact Us
FlyInjection Laboratories 278 Bonny Street Mountain View CA 94043 Tel
888-551-7138 contact@flyinjection.com
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