Service A--Injection only 

We take DNA sample provided by customer, check the concentration.  For p-element injection, DNA is ethanol
precipitated with the helper plasmid (we provide). For phiC31 lines with endogenous integrase source, we dilute
the DNA to the right concentration and inject it directly without precipitation. For attP lines without integrase
source, we will co-inject the DNA with helper integrase DNA(we provide). Prior to injection, we will check DNA
concentration again to make sure enough amounts DNA to be delivered. We use w¹¹¹⁸ and yw as our standard
strains for P-element injection. For phiC31 lines, please see link for details. Please indicate your choice on the
order form, you can download the order form from Place Order section.

We inject 250 to 300 embryos for P-element service and 150 to 200 embryos for phiC31 service.  The  survival
larvae will be sent back to our customer with injection service report.  You are  responsible for identifying the
transformants.

Service B--Injection and screening

We will rear the injected larvae to adulthood in our lab, following the standard microinjection services A. Cross
the G₀ injected flies to W¹¹¹⁸ or yw flies (we usually set up 80 to 100 crosses using single injected male fly cross
to three w¹¹¹⁸ /yw virgins and 2-3 injected virgins cross to three w¹¹¹⁸ /yw male flies) for  P-element injection
and 20 to 50 crosses depending on the number of flies eclosed from injection. Identify the transformants after 10
to 14 days and collect transformant flies in new vials. Only collected transformant flies will be sent to our customer
and original vials will be maintained in our lab for two weeks. 

Timeline for Microinjection Process (Service A)

We work closely with our customers to inform them of our progress and timeline.   The schedule for microinjection
process is:

v     Samples are processed within 1 to 2 days upon arrival.

v     Injection is performed within 2  to 10 days, depending on the workload .

v     Survival larvae are picked in 36 hours after injection.

v     The vials are kept in our incubator for 5 to 7 days before delivery.

Timeline for Microinjection Process and transgenic lines production (Service B)

DNA Requirements

It is critical for the quality and purity of DNA for the successful injection and transformation. We will check the DNA quality 
when sample arrive at our facility. In order to obtain consistent results, we recommend using Qiagen Midi or Maxi column 
for preparing the DNA with size less than 15kb and Invitrogen Purelink Hipure Plasmid Filter Maxiprep kit for large DNA 
construct (size over 20kb). The concentration of DNA is required to be higher than 0.3ug/ul for P-element injection and 
1.0 ug/ul for Phi-C31 line injection. Please dissolve DNA in DEPC treated water for PhiC31 service.

Please provide 35-50ug total DNA in 1.5ml microtube, seal the tube properly with parafilm to prevent cap from poping up 
during the transportation. If the DNA is not enough, we will request more DNA. However, it will delay the service. Here is 
a modified protocol using QIA filter plasmid  Midi Kit (25) (cat no. 12243), it usually gives 100ug to 150ug total DNA from a 
100 ml culture. This method is fairly rapid, and gives high enough quality of DNA for microinjection. 

Grow a 100 ml culture overnight at 37oC shaker incubator. 
Spin down at 5K for 15 minutes. 
Remove all the supernatant 
Resuspend pellet in 8ml buffer "P1". 
Add 8ml P2 to each tube and immediately but gently mix by inversion. Leave in RT for 5 minutes. Do not 
exceed 5 minutes. 
Add 8ml P3 and mix by inversion until solution is clear with floating white stuff. 
Spin down by 10 minutes in the same bottle at 5K, this help to remove most debris and help prevent clog 
the filter column. 
Equilibrate a Tip-100 column by running 5 ml of QBT over it. 
Load the entire supernatant into filter column and filter all the supernatant into the balanced Tip-100 column. 
Wash with 2X 20 ml QC buffer. 
Elute into a15ml culture tube with 8 ml QF buffer. 
Add 5.6ml isopropanol, mix, and centrifuge 30 minutes at 9K to pellet the DNA. For high copy plasmids, the 
DNA pellet should be visible. 
Wash with 70% EtOH, dry 10minutes in 37oC incubator, and resuspend in 500 µl TE.