PCR analysis of DNA from Laser Microdissected (LM) Samples - Embryo Genotyping Protocol
Use sterile conditions through out protocol.
Sterilize the Arcturus heat block and amber colored tray with RNase Zap (Ambion, cat# AM9780) and rinse with Nuclease-free water. Blot with Kim wipes and set under hood to dry.
Set LM lab oven at 65°C. Once up to temperature, place sterile Arcturus heat block in oven with holes up.
Fit Heater/shaker with 500 µl tube holder, turn on and set to 95°C to preheat.
Reagent Preparation
Slide Preparation
LM Staining
Laser Microdissection
DNA Isolation
Prepare all reagents fresh using RNase-free conditions.
Cresyl Violet Acetate Staining-
Use disposable mTubs (Erie Scientific cat# TR-MTUB) during staining for all reagents except stain and xylene steps.
75% Ethanol in nuclease-free water- 400 ml (Use sterile 750 ml plastic flask)
300 ml Absolute Ethanol
100 ml Nuclease-free water
95% Ethanol in Nuclease-free water- 200 ml (Use sterile 250 ml plastic flask)
190 ml Absolute Ethanol from fresh bottle.
10 ml Nuclease-free water
100% Ethanol from fresh bottle.
1% Cresyl Violet acetate (CVa) 50 ml
0.5 gram Cresyl Violet acetate (Sigma-Aldrich, cat# C1791)
50 ml Nuclease-free water in sterile conical tube.
Vortex tube with stain for 15 seconds and set aside to settle.
Sterilize a glass stain dish with Rnase Zap and rinse with nuclease free water. Blot with Kim wipes and place under the hood to completely dry. Use this dish for the xylene steps during deparaffinization.
Just before staining, pull 10 cc of 1% CVa into a sterile syringe.
Fit syringe tip with a 0.22 µm filter (Millipore, cat# SLGS033SS)