Genotyping Protocols
Each genotyping reaction is a little bit different, requiring slightly different conditions for optimum PCR amplification.
The following worksheets show the various conditions that have been optimized for genotyping different mouse lines. Typically, more than one mouse is genotyped at a time. It is easiest to prepare a Master Mix (MM) that contains everything but the DNA for the PCR reactions.
The worksheets show the amount for one reaction.
- If you have five mice to genotype, then multiply the µL amounts for each reagent by nine:
- Five genotyping reactions
- One known positive control
- One negative control with wild type mouse DNA
- One negative control with water
- One extra to compensate for pipetting errors
- Pipet 1 µL of mouse DNA (See the protocol for extracting DNA from mouse tails and yolk sacs.) into the bottom of PCR tubes – one tube per reaction
- Then, aliquot the well-mixed MM into each tube
- For 15 µL reactions, it is not necessary to vortex the reactions and spin them down before putting them into the PCR machine
- For 50 µL reactions, it is advisable to mix each tube thoroughly and spin them down before putting them in the PCR machine
- Primers are used at 10 µM concentration. dNTPs are used at 10 mM concentration
- The H2O is endonuclease-free
- The buffer and MgCl2 used are supplied with the different types of Taq polymerase. These are used without dilution
- All values in the worksheets are in µL
- The MM Volume is the amount to aliquot into each reaction tube
Gcn5 wt/flox
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
WL20-EJ: 0.3 µL - Primer 2
WL21-3-EJ: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
X 9
Five Mice to Genotype
- H2O: 86.85 µL
- Buffer: 13.5 µL
- MgCl2: 4.05 µL
- DMSO: 5.4 µL
- Taq,
Denville Chroma: 8.1 µL - dNTPs: 2.7 µL
- Primer 1
WL20-EJ: 2.7 µL - Primer 2
WL21-3-EJ: 2.7 µL - MM Volume: 126 µL
- DNA: 1 µL
Gcn5 ∆3-18 and Flox(neo)
Gcn5 ∆3-18
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
WL20-EJ: 0.3 µL - Primer 2
WL23-EJ: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gcn5 Flox(neo)
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
WL20-EJ: 0.3 µL - Primer 2
WL21-2-EJ: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gcn5 HAT1 wt and mut (exon 12)
Gcn5 HAT1 wt
(exon 12)
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
JZ-HAT1-wt-F: 0.3 µL - Primer 2
JZ-HAT1-R: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gcn5 HAT1 mut
(exon 12)
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
JZ-HAT1-m-F: 0.3 µL - Primer 2
JZ-HAT1-R: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gcn5 HAT2 wt and mut (exon 13)
Gcn5 HAT2 wt
(exon 13)
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
JZ-HAT2-wt-R: 0.3 µL - Primer 2
JZ-HAT2-F: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gnc5 HAT2 mut
(exon 13)
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
JZ-HAT2-m-R: 0.3 µL - Primer 2
JZ-HAT2-F: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Gcn5 Null
1X MM
- H2O: 9.65 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
Gcn5Df: 0.3 µL - Primer 2
Gcn5Dr: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
PCAF - WT and Null
PCAF - WT
1X MM
- H2O: 3.35 µL
- Buffer D: 7.5 µL
- Taq
Epicentre FailSafe: 0.15 µL - Primer 1
PCAF-WT-F: 1.5 µL - Primer 2
PCAF-WT-R: 1.5 µL - MM Volume: 14 µL
- DNA: 1 µL
PCAF - Null
1X MM
- H2O: 3.35 µL
- Buffer D: 7.5 µL
- Taq
Epicentre FailSafe: 0.15 µL - Primer 1
PCAF-null-F: 1.5 µL - Primer 2
PCAF-null-R: 1.5 µL - MM Volume: 14 µL
- DNA: 1 µL
PCP2-Cre and P300
PCP2-Cre
1X MM
- H2O: 4.29 µL
- Buffer: 1.2 µL
- MgCl2: 0.96 µL
- DMSO: 1.0 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.25 µL
- Primer 1
Cre-For: 0.6 µL - Primer 2
Cre-Rev: 0.6 µL - Primer 3
(IL2 internal control)
Pcp2Con-f: 0.6 µL - Primer 4
(IL2 internal control)
Pcp2Con-R: 0.6 µL - MM Volume: 11 µL
- DNA: 1 µL
P300
1X MM
- H2O: 9.45 µL
- Buffer: 1.5 µL
- MgCl2: 0.45 µL
- DMSO: 0.6 µL
- Taq,
Denville Chroma: 0.9 µL - dNTPs: 0.3 µL
- Primer 1
CS-1: 0.3 µL - Primer 2
PGK-1: 0.3 µL - Primer 3
(Gcn5 internal control)
WL20-EJ: 0.3 µL - Primer 4
(Gcn5 internal control)
WL21-3-EJ: 0.3 µL - MM Volume: 14 µL
- DNA: 1 µL
Band Sizes
This chart shows the expected band sizes for each set of primers:
Genotype
- GCN5 wt
- GCN5 ∆3-18
- GCN5 Flox
- GCN5 Flox(neo)
- GCN5 HAT 1-wt
- GCN5 HAT 1-mut
- GCN5 HAT 2-wt
- GCN5 HAT 2-mut
- GCN5 Null
- P300
- PCP2-Cre
- PCAF-wt
- PCAF-null
Band Size, bp
- 210
- 240
- 310
- 410
- 360
- 360
- 370
- 370
- 450
- 400 bp
210-bp internal control (Gcn5) - 100 bp
320-bp internal control (IL2) - 175
- 390
| Gene | Primer Name | Sequence |
|---|---|---|
| Gcn5 WT | WL20-EJ | CACAGAGCTTCTTGGAGACC |
| Gcn5 WT | W 21-3-EJ | GGCTTGATTCCTGTACCTCC |
| Gcn5 Δ3-18 | WL23-EJ | ATAGTAGCGACTGCGCAACC |
| Gcn5 Flox(neo) | WL21-2-EJ | CTGTGCCTTCTAGTTGCCAG |
| Gcn5 HAT1 WT exon 12 | JZ-HAT1-wt-F | TTCCCACTCAGGGCTTCACGGA |
| Gcn5 HAT1 exon 12 | JZ-HAT1-R | CTCTCACAGTAGACCAGGCTGG |
| Gcn5 HAT1 mut exon 12 | JZ-HAT1-m-F | CCCACTCAGGGCTTCACGCG |
| Gcn5 WT exon 13 | JZ-HAT2-wt-R | TGAAGTAGCCAATGGCGTACTCGT |
| Gcn5 exon 13 | JZ-HAT2-F | GTGGTCCACTGACCTCAGACTG |
| Gen5 mut exon 13 | JZ-HAT2-m-R | TGAAGTAGCCAATGGCGTACTCTG |
| Gcn5 null | Gcn5Df | TCACTATCTCGGATGGCTT |
| Gcn5 null | Gcn5Dr | CCTCTTCGCTATTACGCCAG |
| PCAF WT | PCAF-WT-F | AAGCTGCTCACGTTTCTCACTTGG |
| PCAF WT | PCAF-WT-R | GGGTAAGCTCTCACCTTGAATAGG |
| PCAF null | PCAF-Null-F | GGATGTGGAATGTGCGAG |
| PCAF null | PCAF-Null-R | TCCAACAGCCTCTCTAGCCT |
| IL2 WT | Pcp2Con-f | CTAGGCCACAGAATTGAAAGATCT |
| IL2 WT | Pcp2Con-r | GTAGGTGGAAATTCTAGCATCATCC |
| Pcp2 | Cre-For | GCGGTCTGGCAGTAAAAACTATC |
| Pcp2 | Cre-Rev | GTGAAACAGCATTGCTGTCACTT |
| p300 | CS-1 | CATGCTCACAAGTGCCAGC |
| p300 | PGK-1 | CTGCACGAGACTAGTGAGACG |
All the above protocols were optimized for the following PCR program:
| Cycles | 1 | 15 | 25 | 1 | Hold | ||||
|---|---|---|---|---|---|---|---|---|---|
| Temp (°C) | 95 | 94 | 65 | 72 | 94 | 50 | 72 | 72 | 4 |
| Time | 5:00 | :30 | :30 | 1:30 | :30 | :30 | 1:30 | 10:00 | Hold |