Protocol for 16S PCR, SAP/Exo, Sequencing Reaction

Dilute primers to 100 uM by adding 10x # of nmol H2O
Working stock (5 uM): 2.5uL stock, 47.5uL dH2O
PCR
MM1
Comp
1x Volume
40x
Final Conc
Water
6 μl
240
dNTPs
.5 μl
20
200 μM
R primer
2.5  μl
100
0.5 μM
F primer
2.5 μl
100
0.5 μl
MM2
Water
9.17 μl
366.8
10x buffer
3.2 μl
128
2 mL MgCl2
Taq
.13 μl
5.2
1.25 U/rxn
Add 1 μl DNA to each tube
Step
Cycles
Time
Temp
Initial Denaturation
1
2 min
94 deg
Denaturation
Annealing
Elongation
1-8
30 s
1 min
1 min
94
54,53,52,51,50,49,48,48
72
Denaturation
Annealing
Elongation
9-33
30 s
1
1
94
48
72
Final Elongation
1
7
72
forever
10
SAP/Exo
MM
Reagent
1x
13x
Sap
0.5 ul
6.5
Exo
0.5 ul
6.5
5 ul of PCR product + 1uL MM
Step
Time
Temp
Incubate
30 min
37 C
Denature
15 min
80 C
Chill
forever
25 C
Sequencing
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2 thoughts on “Protocol for 16S PCR, SAP/Exo, Sequencing Reaction

  1. Pingback: Wed. Oct. 22, 2014 | Getting My Genes Wet

  2. Pingback: Tues. Nov. 4, 2014 | Getting My Genes Wet

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