Genotyping
AC1 TG mouse
Primers:
INT265: ATC GAT TCT AGA ATT CGC TGT CTG
FLAGR: CTT ATC GTC GTC ATC CTT GTA ATC
For each 20 ul reaction:
12 ul H20
4 ul Buffer Q (Qiagen)
2 ul 10 X PCR buffer (traditional, and without any dye)
0.125 ul dNTP (25 mM)
0.5 ul INT265 (10 uM or 10 pmole/ul)
0.5 ul FLAGR (10 uM)
Mix well and add 0.1 ul Taq (Qiagen).
PCR Program:
1. 94 degrees for 3 min
2. 94 degrees for 30 sec
3. 58 degrees for 30 sec
4. 72 degrees for 45 sec
5. Go to step 2 and repeat 35 times
6. 72 degrees for 5 min
7. Hold at 4 degrees
Expected size: 340 bp
AC1 mouse
Primers:
AC1Up: CTT TCC CTG GCG CTG TTC GTG
AC1Down: CTG TGA CCA GTA AGT GCG AGG
NeoUp: GTC ATC TCA CCT TGC TCC TGC
NeoDown: GGT AGC CAA CGC TAT GTC CTG
For each 20 ul reaction:
13.775 ul H20
0.125 ul dNTP (25 mM)
4 ul 5 x PCR buffer
0.5 ul AC1Up (20 uM)
0.5 ul AC1Down (20 uM)
0.5 ul NeoUp (20 uM)
0.5 ul NeoDown (20 uM)
0.1 Taq
PCR Program:
1. 95 degrees for 3 min
2. 95 degrees for 1 min
3. 55 degrees for 1 min
4. 72 degrees for 1 min
5. Repeat Steps 2-4 35 times
6. 72 degrees for 7 min
7. Hold at 4 degrees
Expected Size: 300 bp AC1, 370 bp neomycin
AC8 mouse
Primers:
PGK2: GGT GGA TGT GGA ATG TGT GC
KO3.1: GTT CAG ACA TCT GTG TCC AC
AD8-10: CGC AGA TCA CCA CCT CGA T
AD8-11: CTG CCT CTC TAT TCT CTG G
For each 20 ul reaction:
15 ul H20 0.16 ul dNTP (25 mM)
4 ul 5 X PCR buffer
0.2 ul PGK2 (30 uM)
0.2 ul KO3.1 (30 uM)
0.06 AD8-10 (100 uM)
0.06 AD8-11 (100 uM)
0.1 ul Taq
PCR Program:
1. 94 degrees for 3 min
2. 94 degrees for 1 min
3. 55 degrees for 1 min
4. 72 degrees for 2 min
5. Repeat steps 2-4 35 times
6. 72 degrees for 7 min
7. Hold at 4 degrees
Expected size: 400 bp WT, 290 bp mutant
FE65 mouse
Primers:
230-199R: GCT TGA GTC CTC ATC ACT G
mFE65wt-F: CTT GAT CAT CAA CTC CCA GG
neo-4F: CGC CTT CTT GAC GAC TTC
For each 19 ul reaction:
2 ul 10 X PCR buffer
2 ul primer mix (10 pmol/ul each)
0.1 ul dNTP (25 mM)
0.1 ul Biolase
13.3 ul H2O
Note: final Mg+2 concentration should be 1.5 mM. Adjust if necessary.
PCR Program:
1. 94 degrees for 4 min
2. 94 degrees for 30 sec
3. 62 degrees for 30 sec
4. 72 degrees for 30 sec
5. Repeat steps 2-4 30 times
6. 72 degrees for 5 min
7. Hold at 4 degrees
Expected Result: 398 bp KO, 258 bp WT
CAMK4
Primers:
oIMR3406: TCA ATT GAA AAG TTC ACT GGA AGA
oIMR3407: TTT GAG AGC ATA GGG CTT CTG
oIMR6916: CCT GGG TGG AGA GGC TAT TC
oIMR6917: AGG TGA GAT GAC AGG AGA TC
For each 20 ul reaction:
13 ul H20
4 ul 5 X Green Buffer
0.15 ul dNTP (25 mM)
0.3 ul 6916 primer
0.3 ul 6917 primer
0.6 ul 3406 primer
0.6 ul 3407 primer
0.1 ul Taq
1 ul DNA (from mouse tail)
PCR Program:
1. 94 degrees for 3 min
2. 94 degrees for 30 sec
3. 62 degrees for 1 min
4. 72 degrees for 45 sec
5. Repeat steps 2-4 35 times
6. 72 degrees for 5 min
7. Hold at 4 degrees
Expected results: 280 bp mutant, 150 bp WT
Note: FE65 shows 250 bp band, can't be used as perfect WT control.
FMR mouse
Primers:
FMRmutant: CAC GAG ACT AGT GAG ACG TG
FMRWT: TGT GAT AGA ATA TGC AGC ATG TGA
FMRcommon: CTT CTG GCA CCT CCA GCT T
For each 20 ul reaction:
4 ul 5 X buffer
0.2 ul dNTP (25 mM)
2.4 ul FMRcommon (20uM)
1.2 ul FMRWT (20 uM)
2.4 ul FMRmutant (20 uM)
0.2 ul Taq
11 ul ddH2O
1 ul DNA
PCR Program:
1. 2. 3. 4. 5. 6. 7.
Expected results:
PKCa mouse
Primers:
240: GCG CAT CGC CTT CTA TCG C
739: AGC TAG GTC CTG TTG GTA AC
1376: CCA AGT GTG AAG TGT GTG AG
For each 20 ul of reaction:
14.275 ul dH2O
0.125 ul dNTP (25 mM)
4 ul 5X buffer
0.5 ul 240 primer (20 mM)
0.5 ul 739 primer (20 mM)
0.5 ul 1376 primer (20 mM)
0.1 ul Taq
PCR Program:
1. 94 degrees for 3 min
2. 94 degrees for 30 sec
3. 57 degrees for 1 min
4. 72 degrees for 1 min
5. Repeat steps 2-4 30 times
6. 72 degrees for 5 min
7. Hold at 4 degrees
Expected results:
CRND8 mice
Primers:
Primer 1: GGC CGC GGA GAA ATG AAG AAA CGC CAA GCG CCG TGA CT
Primer 2: TGT CCA AGA TGC AGC AGA AVG GVT ACG AAA A
For each 20 ul of reaction:
8.8 ul ddH2O
4 ul 5X Q buffer
2 ul 10X buffer
2 ul dNTP
1 ul primer 1 (20 mM)
1 ul primer 2 (20 mM)
0.2 ul Taq
PCR Program:
1. 94 degrees for 3 min
2. 94 degrees for 20 sec
3. 68 degrees for 20 sec
4. 72 degrees for 90 sec
5. Repeat steps 2-4 35 times
6. 72 degrees for 7 min
7. Hold at 4 degrees
Keywords
Adenylyl cyclase (AC), amyloid precursor protein (APP), Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), cAMP, cAMP-responsive element (CRE), CREB, extra-cellular regulated protein kinase (ERK), FE65, long-term potentiation (LTP), long-term depression (LTD), memory formation, neuroplasticity, local protein synthesis.