imported>Virgilio |
imported>Xenbase |
Line 1: |
Line 1: |
| Protocol submitted by VGP from Stukenberg lab protocols [http://www.xenbase.org/community/person.do?method=display&personId=1311&tabId=0]
| | ==Reporting a bug in Xenbase== |
| | If you find a bug in Xenbase, we would really like to know! |
| | However, it is very important that we can find your bug so that we can fix it. |
| | To help us find your bug, please fill out [http://xenbase.org/xenwiki/images/6/6e/Bug_report.txt this] form, and e-mail it to us! |
|
| |
|
| | | ==Bug Handling Process== |
| link to protocol page [http://people.virginia.edu/~djb6t/LabWeb/frogs.htm]
| |
| | |
|
| |
| Sara's Cycling Xenopus Extracts
| |
|
| |
| | |
| To prepare: 5 days before the experiment, prime female frogs with 50 IU of PMSG.
| |
|
| |
| The night before the experiment, induce ovulation by injecting the primed frogs with 500 IU of HCG. Place each frog in 2L of 1X MMR overnight at 18°C.
| |
|
| |
| In the morning, collect laid eggs. Place the frogs back into 1X MMR and continue to collect eggs.
| |
|
| |
| Once you have sufficient amounts of eggs, wash the eggs in 1X MMR to remove debris and bad eggs.
| |
|
| |
| Remove as much MMR as possible and de-jelly the eggs with 2% cysteine solution prepared in 1X XB salts. Swirl eggs occasionally and de-jelly until the eggs pack tightly. Pour off de-jelly, and rinse briefly with fresh de-jelly solution.
| |
| | |
| Wash the eggs into 1X MMR. Activate the eggs by addition of 2μg/mL A23187, a calcium ionophore. (To 10 mL MMR, add 1μL ionophore). Watch for activation (takes about 10 minutes). Upon activation, wash out ionophore liberally with MMR. Place the eggs at 18°C for 50 minutes.
| |
|
| |
| Wash the eggs 4X with XB, and 2X with XB+protease inhibitors (10μg/mL each leupeptin, pepstatin and chymostatin (LPC)). Load the eggs gently into ultraclear centrifuge tubes containing 1 mL XB+PIs and 100μg/mL cytochalasin B or D.
| |
|
| |
| Pack the eggs by spinning at low speed in a clinical centrifuge for 30-60 seconds. Aspirate off the buffer and spin at moderate speed in the clinical centrifuge for 30-60 seconds. Aspirate off residual buffer.
| |
|
| |
| Crush eggs by spinning in the mini ultracentrifuge swinging bucket rotor (RP55S-344) at 10,000 rpm for 10 minutes at 2°C.
| |
|
| |
| Remove the cytoplasmic fraction with a 1mL syringe and a 18-guage needle.
| |
|
| |
| Add 1/1000 volume of LPC, 1/1000 volume of cytochalasin B or D, and 1/40 energy mix and 1/40 sucrose to the cytoplasmic fraction. Invert and flick gently to mix.
| |
|
| |
| The extract is now ready for use.
| |