LDLR Genotyping: Difference between revisions

Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/LDLR_Genotyping"

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-Based on https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075
+Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075
-LDLR Genotyping Common - Fwd 	TAT GCA TCC CCA GTC TTT GG
+* [[SOP - Ethidium Bromide]]
-LDLR Genotyping WT - Rev 	CTA CCC AAC CAG CCC CTT AC
+* [[SOP - Electrophoresis]]
-LDLR Genotyping Mutant  - Rev 	ATA GAT TCG CCC TTG TGT CC
-[[ Genotyping ]]
+== Reagents Needed ==
-[[ Mouse Work ]]
-[[ PCR ]]
+=== PCR Primers ===
+* '''Ldlr Common Forward''': 5'-TAT GCA TCC CCA GTC TTT GG-3'
+* '''Ldlr Wild-type Reverse''': 5'-CTA CCC AAC CAG CCC CTT AC-3'
+* '''Ldlr Mutant Reverse''': 5'-ATA GAT TCG CCC TTG TGT CC-3'
+=== Master Mix ===
+* '''DreamTaq Green PCR Master Mix (2X)''' - Thermo Fisher Scientific, Cat# K1081
+=== Additional Reagents ===
+* Nuclease-free water
+* Template DNA ([[Preparation of Tail Samples (for Genotyping)|tail lysate]] or purified genomic DNA)
+== Primer Preparation ==
+=== Stock Primer Preparation (100 µM) ===
+# Resuspend each lyophilized primer in nuclease-free water to 100 µM concentration.  Look on the tube from IDT, for example if there is 23.7 nmoles then you would resuspend in 237 µL of water
+# Store at -20°C
+=== Primer Master Mix (10 µM each primer) ===
+For a 1 mL primer master mix:
+* 100 µL of 100 µM Primer 19799
+* 100 µL of 100 µM Primer 19800
+* 100 µL of 100 µM Primer oIMR7770
+* 700 µL nuclease-free water
+'''Final concentrations in primer master mix:''' 10 µM each primer
+Store at -20°C
+== PCR Master Mix Preparation ==
+=== Per Reaction (25 µL total volume) ===
+* 12.5 µL DreamTaq Green PCR Master Mix (2X)
+* 3.75 µL Primer Master Mix (10 µM each)
+* 7.75 µL nuclease-free water
+* 1 µL template DNA
+'''Final primer concentrations in PCR:''' 1.5 µM each primer
+=== For Multiple Reactions ===
+Prepare master mix for (n+1) reactions, where n = number of samples:
+* DreamTaq Green PCR Master Mix (2X): 12.5 µL × (n+1)
+* Primer Master Mix: 3.75 µL × (n+1)
+* Nuclease-free water: 7.75 µL × (n+1)
+Aliquot 24 µL of master mix per tube, then add 1 µL template DNA to each
+== PCR Cycling Program ==
+This program takes 1:50 to run.
+{| class="wikitable"
+|+ '''Ldlr''' PCR Program
+|-
+! Step !! Temperature (°C) !! Time !! Cycles !! Notes
+|-
+| Initial Denaturation || 94 || 3 min || 1 ||
+|-
+| '''Touchdown Phase''' || || || ||
+|-
+| Denature || 94 || 30 sec || rowspan="3" | 10 || rowspan="3" | Decrease annealing temp by 0.5°C per cycle
+|-
+| Anneal || 65→60 || 30 sec
+|-
+| Extend || 68 || 45 sec
+|-
+| '''Standard Cycles''' || || || ||
+|-
+| Denature || 94 || 30 sec || rowspan="3" | 28 ||
+|-
+| Anneal || 60 || 30 sec ||
+|-
+| Extend || 72 || 45 sec ||
+|-
+| Final Extension || 72 || 5 min || 1 ||
+|-
+| Hold || 4 || ∞ || ||
+|-
+|}
+== Expected Results ==
+* '''Wild type:''' 351 bp
+* '''Heterozygote:''' 179 bp AND 351 bp
+* '''Mutant:''' 179 bp (GC-rich band)
+'''Note:''' Run on 2% agarose gel. The mutant band (179 bp) is GC-rich and may appear fainter than expected.
+[[ Category:Genotyping ]]
+[[ Category:Mouse Work ]]
+[[ Category:PCR ]]