LDLR Genotyping: Difference between revisions
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Modifieed protocol from Jax, as modified by Claude to use dreamtaq |
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Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075 | |||
== Reagents Needed == | |||
=== PCR Primers === | |||
* '''Primer 19799''' (Common Forward): 5'-TAT GCA TCC CCA GTC TTT GG-3' | |||
* '''Primer 19800''' (Wild type Reverse): 5'-CTA CCC AAC CAG CCC CTT AC-3' | |||
* '''Primer oIMR7770''' (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 (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 | |||
# 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 == | |||
{| class="wikitable" | |||
|+ Ldlr Genotyping 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 || 10 || ∞ || || | |||
|- | |||
|} | |||
== 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:Genotyping ]] | ||
[[ Category:Mouse Work ]] | [[ Category:Mouse Work ]] | ||
[[ Category:PCR ]] | [[ Category:PCR ]] | ||
Revision as of 19:33, 24 January 2026
Modified from https://www.jax.org/Protocol?stockNumber=002207&protocolID=27075
Reagents Needed
PCR Primers
- Primer 19799 (Common Forward): 5'-TAT GCA TCC CCA GTC TTT GG-3'
- Primer 19800 (Wild type Reverse): 5'-CTA CCC AAC CAG CCC CTT AC-3'
- Primer oIMR7770 (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 (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
- 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
| Step | Temperature (°C) | Time | Cycles | Notes |
|---|---|---|---|---|
| Initial Denaturation | 94 | 3 min | 1 | |
| Touchdown Phase | ||||
| Denature | 94 | 30 sec | 10 | Decrease annealing temp by 0.5°C per cycle |
| Anneal | 65→60 | 30 sec | ||
| Extend | 68 | 45 sec | ||
| Standard Cycles | ||||
| Denature | 94 | 30 sec | 28 | |
| Anneal | 60 | 30 sec | ||
| Extend | 72 | 45 sec | ||
| Final Extension | 72 | 5 min | 1 | |
| Hold | 10 | ∞ |
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.
