Revision as of 00:22, 24 September 2016 by >Djacobse
PCR Testing (Started Yesterday)
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Protocol (all)
- Circularization
- Heat inactivate enzyme by incubating for 2 minutes at 94C
- Exonuclease Digestion
- Prepare exonuclease I/III mix by mixing 20 uL exonuclease I (20 units/μL) and 4 uL exonuclease III (100 units/μL) in 1:1 ratio
- Add 2 μL of exonuclease I/III mix to reaction
- Mix the reaction by swirling pipette around the well 5 times
- Incubate reaction at 37 ºC for 2 hours
- Heat inactivate enzyme by incubating at 94C for 5 minutes
Standard qPCR like I normally do. Control version for the tests after.
- qPCR
- Follow reaction map below
- Note:"X"=5 uL primer per reaction; "O" indicates 1 uL primer
- Note:"TN" is a range of temperatures (gradient) from 50C (Lane 1) to 65C (Lane 12)
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1
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2
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3
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4
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5
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6
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7
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8
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9
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10
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11
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12
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A
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X,T1
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X,T2
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X,T3
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X,T4
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X,T5
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|
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X,T6
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B
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X,T1
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X,T2
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X,T3
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X,T4
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X,T5
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|
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X,T6
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C
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X,T1
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X,T2
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X,T3
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X,T4
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X,T5
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|
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X,T6
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D
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O,T1
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O,T2
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O,T3
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O,T4
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O,T5
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|
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O,T6
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E
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O,T1
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O,T2
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O,T3
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O,T4
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O,T5
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|
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O,T6
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F
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O,T1
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O,T2
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O,T3
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O,T4
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O,T5
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|
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O,T6
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G
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NTC
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NTC
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NTC
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NTC
|
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NTC
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|
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NTC
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H
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|
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- Set up reaction according to table in next section
- Make master mixes according to following table
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Master Mix
|
Reagent
|
MMX
|
MMO
|
NTC
|
PrimerF (100 uM)
|
9.05
|
1.81
|
3.05
|
PrimerR (10 uM)
|
90.5
|
18.1
|
30.5
|
KAPA Master Mix
|
452.5
|
452.5
|
152.5
|
nfH2O
|
316.75
|
396.39
|
118.95
|
- Add 48 uL correct master mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
qPCR Sample Table
Sample
|
Molecule Count
|
Lane
|
AmpR Index
|
Sample Vol (uL)
|
2X Kapa SYBR qPCR MM
|
100 uM AmpF 6.4
|
10 uM AmpR6.3-IndXX
|
H2O
|
Total Volume (uL)
|
Master Mix
|
Sample 1A
|
1.00E+10
|
A1
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1B
|
1.00E+10
|
B1
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1C
|
1.00E+10
|
C1
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1A
|
1.00E+09
|
D1
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1B
|
1.00E+09
|
E1
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1C
|
1.00E+09
|
F1
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H1
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Sample 1A
|
1.00E+10
|
A3
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1B
|
1.00E+10
|
B3
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1C
|
1.00E+10
|
C3
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1A
|
1.00E+09
|
D3
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1B
|
1.00E+09
|
E3
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1C
|
1.00E+09
|
F3
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H3
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Sample 1A
|
1.00E+10
|
A5
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1B
|
1.00E+10
|
B5
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1C
|
1.00E+10
|
C5
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 1A
|
1.00E+09
|
D5
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1B
|
1.00E+09
|
E5
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 1C
|
1.00E+09
|
F5
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H5
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Sample 2A
|
1.00E+10
|
A7
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2B
|
1.00E+10
|
B7
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2C
|
1.00E+10
|
C7
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2A
|
1.00E+09
|
D7
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2B
|
1.00E+09
|
E7
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2C
|
1.00E+09
|
F7
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H7
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Sample 2A
|
1.00E+10
|
A9
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2B
|
1.00E+10
|
B9
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2C
|
1.00E+10
|
C9
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2A
|
1.00E+09
|
D9
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2B
|
1.00E+09
|
E9
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2C
|
1.00E+09
|
F9
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H9
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Sample 2A
|
1.00E+10
|
A12
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2B
|
1.00E+10
|
B12
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2C
|
1.00E+10
|
C12
|
34
|
2
|
25
|
0.5
|
5
|
17.5
|
50
|
MMX
|
Sample 2A
|
1.00E+09
|
D12
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2B
|
1.00E+09
|
E12
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
Sample 2C
|
1.00E+09
|
F12
|
35
|
2
|
25
|
0.1
|
1
|
21.9
|
50
|
MMO
|
NTC
|
0
|
H12
|
36
|
0
|
25
|
0.5
|
5
|
19.5
|
50
|
NTC
|
Results
Extension Time
- qPCR
- Set up reaction according to table
- Master Mix
- 480 uL KAPA SyberFast MM
- 336 uL nfH2O
- 9.5 uL AmpF 6.4
- Add 43 uL Master Mix to each well
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x20
- 72C 2 min
- 16C hold