Transgene Construct Preparation Protocol
Transgene Preparative Restriction Digest – 2 enzymes
Digest: 40 ul total volume (do 6 digests)
_______ ul DNA prep______(~3ug)
_______ ul H20
4 ul 10X buffer _________
4 ul 10X BSA
1.5 ul of each enzyme__________
If using 1 enzyme, use 3ul of enzyme per restriction digest
Incubate minimum 4 hours, 37C (note if different temp required – check enzyme list)
Run on 1% low melt gel (100ml) overnight, 30V, 20ul of ladder
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands and place in microtubes – hold at 4C until next gel is ready
Pour 1% low melt midi-gel (100ml) without comb, check the gel 5 minutes after pouring should not set-up more than 7 - 10 minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (~ 1 hour)
Run overnight 30V (= 2nd overnight)
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands and place in microtubes – hold at 4C until next gel is ready
Pour 1% low melt midi-gel (100ml) without comb, check the gel 5 minutes after pouring should not set-up more than 7 - 10 minutes before adding cut gel bands
Place gel bands into new gel and let stand RT until completely set up (~ 1 hour)
Run overnight 30V (= 3rd overnight)
Let run all day the next day at 30V – DO NOT turn up the voltage
Cut gel bands, store 4C or proceed with Qiaex II purification
Qiaex II purify – ELUTE with transgene injection buffer
Transgene Injection Buffer
Tris (pH 7.4): 10mM
NaCl: 10mM
EDTA: 0.25mM
OD260/280: concentration_____________ ratio:______________
Run 100ng on 1% gel to check – take 2 photos – 1 for your records and 1 for the MGC
*NOTE: For BAC Transgenes please contact Mia (mia@wustl.edu) for the BAC preparation protocol.
ES Cell Preparation for Blastocyst Injections
Help with designing your targeting vector and embryonic stem cell transfection service is offered by the Siteman Cancer Center Stem Cell Core. The MGC will coordinate arrangements with the Stem Cell Core to prepare and transfer your targeted cells on injection days. If you provide your own cells, please use the following protocol to prepare them for injection:
Thaw cells from appropriate positive clone or clones onto a 6cm dish plated with a MEF feeder layer at least 3-5 days prior to injection dates depending on growth speed of your cells. Split cells just before they become confluent, usually 2-3 days before injections, onto three 6cm plates – one for each day of injections as follows: 50% for injection day 1; 30% for day 2; and 20% for day 3.
On the morning of the injections, feed the cells two hours prior to injection time. Just before the scheduled time of transfer, trypsinize the cells for five minutes with pre-warmed trypsin at 37ºC. Place the trypsinized cells in a 15 mL tube, and add an equal volume of ES cell media to inactivate the trypsin. Centrifuge the cells 100-200 x g and remove the supernatant. Resuspend the cells in 1mL of 'injection media' (see below) + 10% FBS. Prepare a second tube containing 10mL injection media + 10% FBS for use on our injection slides. Complete separation of ES cells is essential for blastocyst injections - please GENTLY pipette cells 30-40 times to ensure cell separation.
Bring the cell suspension and extra injection media to us on wet ice.
Injection Media: (available through the tissue culture center on campus)
DME
25mM HEPES without NaHCO3
Osmolarity = 290
PH = 7.4
Universal Mouse Genotyping
PCR primers are required for all new transgenic constructs or requests for genotyping services. These primers should be 30 nucleotides in length and produce an amplimer of between 100 and 500 nucleotides. Please see our list of primer pairs that work well for common sequences.
This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon, Transgenic Research 12, 521-522 (2003). Materials and procedures are described in complete detail.