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In Situ Hybridization (view source)
Revision as of 11:26, 30 September 2022
, 11:26, 30 September 2022added buffer amounts
'''DTT (dithiothreitol)'''
'''DTT (dithiothreitol)'''
Aka Cleland's reagent; prevents oxidation of thiol groups and breaks down disulfide bonds.
Aka Cleland's reagent; prevents oxidation of thiol groups and breaks down disulfide bonds.
==Buffer Amounts==
* Denature salmon sperm by boiling for 10 minutes.
* Put DTT in foil-covered scintillation vial.
* Add SSC, formamide, TED, salmon sperm, & Denharts solution.
* Vortex. Keep heated at 55° C on heater block until use.
<tab class="wikitable" sep="tab" border = "1" cellspacing="3" cellpadding = "3" head = top>
10 ml - - - 8 ml - - - 5 ml - - - 4 ml - - - 3 ml - - - 2 ml - -
150 mg DTT 120 mg DTT 75 mg DTT 60 mg DTT 45 mg DTT 30 mg DTT
1.0 ml SSC 20x 800 ml SSC 20x 500 µl SSC 20x 400 µl SSC 20x 300 µl SSC 20x 200 µl SSC 20x
6.0 ml Formamide 4.8 ml Formamide 3.0 ml Formamide 2.4 ml Formamide 1.8 ml Formamide 1.2 ml Formamide
2.0 ml TED 5x 1.6 ml TED 5x 1 ml TED 5x 800 µl TED 5x 600 µl TED 5x 400 µl TED 5x
1.6 ml Salmon Sperm 1.28 ml Salmon Sperm 800 µl Salmon Sperm 640 µl Salmon Sperm 480 µl Salmon Sperm 320 µl Salmon Sperm
800 µl Denharts 50x 640 µl Denharts 50x 400 µl Denharts 50x 320 µl Denharts 50x 240 µl Denharts 50x 160 µl Denharts 50x
</tab>