GENOTYPING BY PCR PROTOCOL FORM

GENOTYPING BY PCR PROTOCOL
MUTANT MOUSE REGIONAL RESOURCE CENTER: UC DAVIS
[email protected]
530-754-MMRRC
NAME OF PCR: Universal tdTomato
MMRRC # UCD
Protocol:
Reagent/ Constituent
Water
10x Buffer
MgCl2
(stock concentration is 25mM)
Betaine (stock concentration is 5M)
dNTPs (stock concentration is 10mM)
DMSO
Primer 1 (stock concentration is 20μM) tdTomato Fwd
Primer 2 (stock concentration is 20μM) tdTomato Rev2
Taq Polymerase (5Units/μL)
DNA extracted with
NaOH
Proteinase K
Volume (μL)
11.725
2.5
1.7
6.5
0.5
0.325
0.25
0.25
0.25
1.0
Other:
TOTAL VOLUME OF REACTION:
25.00
Comments on protocol:
o
o
Use Touch-Down cycling protocol-first 10 cycles anneal at 65 C decreasing in temperature by 1.0 C; next 30 cycles
o
anneal at 55 C.
Betaine/DMSO is standardized due to high GC content in promoter regions and protocol may be tested without. Also, may
adjust MgCl2 to increase reaction or decrease non specific amplifications.
Strategy:
Steps
1. Initiation/Melting
2. Denaturation
3. Annealing
4. Elongation
5. Amplification
6. Finish
}
o
HOT START?
steps 2-3-4 will cycle in sequence
Temp ( C )
94
94
65 to 55 (↓1oC/cycle)
72
72
4
Time (m:ss)
5:00
0:15
0:30
0:40
5:00
# of Cycles
∞
n/a
Primers:
Name
Nucleotide Sequence (5' - 3')
AGCAAGGGCGAGGAGGTCATC
CCTTGGAGCCGTACATGAACTGG
1: tdTomato Fwd
2: tdTomato Rev2
Electrophoresis Protocol:
Agarose: 1.5%
V: 90
Estimated Running Time: 90
min.
Primer Combination
Band
Genotype
1 and 2
208 bp
tdTomato
1
2
3
4
Lanes
1. No Template Control
2. Wild-type
3. tdTomato positive
4. tdTomato positive
PCR protocol developed by MMRRC at University of California, Davis
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