Q: What is the turn-around time in
general?
A: The turn-around time currently is about 10 to
14 days after we receive your sample. The injection is typically
processed in 3 to 7 days; however, we will observe the injected
larvae at 25oC for 5 to 7 days before we send them back.
The latest turn-around time is posted on Place
Order page.
Q: What is the best way to send in DNA?
Do I need ice or dry ice?
A: It is not necessary to use ice or dry ice to
send your DNA. Most people use FedEx, UPS or DHL express to send in
their DNA. We recommend using the Second Day Air to avoid
degradation of the DNA during transportation. We check the DNA
concentration before injection and will inform you if there is any
problem.
Q: How do you send back the injected
sample?
A: We individually pack the samples in our lab.
The package is shipped using FedEx Priority Overnight or USPS
express mail (within US), and FedEx priority (overseas). An
email notice regarding the shipping and tracking information is sent
out immediately the package is shipped out. We are not responsible
for any transportation caused damage, however, we may provide a
re-injection without additional charge.
Q: What is the survival rate of
injection? How many independent transformant lines can I
get?
A: It depends on a few factors, for example, the
DNA quality, the DNA expression level and the insertion size, etc.
For P-element construct, an injection of 250 embryos normally
yields 150 to 200 survival larvae, of which 50-70% will develop into
pupae. You can then get 10 to 20 independent transformant
lines.
Q: How do we prepare our DNA? What commercial column
should we use?
A: DNA quality is very
important for successful injections. 95% injections fail due to poor
DNA quality. We strongly
recommend you use Qiagen Midi or Maxi kit to prepare your DNA
(size less than 15kb) and Invitrogen
Purelink Hipure
Plasmid Filter Maxiprep kit for large DNA construct preparation
(size over 20kb). Please don't use Qiagen mini column for
preparing DNA for injection.During the preparation, make sure
the column is balanced well and washed thoroughly ( 2 to 3 times)
before eluting the DNA. In our experiences, DNA prepared by
Biorad will kill the embryos; even Phenol/Chloroform extraction
cannot help. Sigma Gene elute HP column needs to be washed many
times.
Q:
How many crosses do we need to set up to get enough
transformants?
A:
Our injection efficiency is consistently high, around 20 to 25%. We
usually send back to customers 150 to 200 survival larvae from which
over 50% to 70% can develop into adult flies. To our experience, 80
crosses are sufficient to give you 10-20 independent transformant
lines for insertion less than 10kb. However, for insertion
size over 10kb or those hard to be transformed, we recommend
you to set up as many crosses as you can.
Q: How do we set up
cross?
A: We recommend
crossing single injected male fly to three w- virgins and
two or three injected virgins to three or four male w-
flies.
Q:
What buffer should I use to send DNA for injection?
A:
For P-element injection, TE or EB buffer can be used to dissolve
your DNA. However, for PhiC31 lines injection, we recommend using
DEPC treated water.
Q:
What the transformation efficiency for PhiC31 lines?
A:
The efficiency is quite high for phiC31 lines, it can reach 30%
to 50% (# of transgenic lines to # of cross). However,
the survival rate is quite low for some lines with endogenous
integrase source.
