SwitchGear Genomics © SwitchGear Genomics

RECOMMENDED GENOMIC DNA AMPLIFICATION PROTOCOL

Round A

Perform two rounds of primer extension using a partially degenerate primer ("primer A"), Sequenase, and slow ramp times

Primer A = 5’ GTTTCCCAGTCACGATCNNNNNNNNN 3’

Step 1: Combine DNA, buffer, and Primer A
DNA (2-400 ng)
15
Sequenase Buffer (5x)
4
Primer A (100 uM)
1
 
20


Step 2: Begin thermalcycler program with 20 ul DNA mix
2 cycles  
98°C
 5 min.
8°C
 4 min. (add reaction mix or diluted Sequenase during last 2 min. of 8°C incubation)
37°C
 slow ramp to 37°C (10% ramp; takes about 6 min.)
37°C
 8 min.


Step 3: Add reaction mix in Cycle 1
Add 10 uL of reaction mix during 8°C inubation for the first round of primer extension
When rxn mix is added, be sure samples are kept at 8 degrees or on ice
 
x10
x25
x30
Sequenase Buffer (5x)
2.00
20
50
60
dNTP mix (25 mM each)
0.36
3.6
9
10.8
DTT (0.1M)
1.00
10
25
30
BSA (10 mg/ml)
0.15
1.5
3.75
4.5
Sequenase (13 U/ul)
.30
3
.75
9
water
6.19
61.9
154.75
185.7
 
10.00
      


Step 4: Add Sequenase in Cycle 2
Add 2 uL of diluted Sequenase during 8°C incubation in the second round of primer extension
When Sequenase is added, be sure samples are kept at 8 degrees or on ice
Sequenase Dilution Buffer
1.7
Sequenase
0.3
 
2


Step 5: Clean up extension reaction
Purify the 32 ul reaction from Round A using QIAgen PCR Cleanup Kit.
Elute twice with 75 ul EB, pool elutions for a total of ~145 uL of cleaned up product


Round B

Perform amlification reaction on DNA from Round A using Primer B

Primer A = 5’ GTTTCCCAGTCACGATCNNNNNNNNN 3’

Step 1: Set up PCR reaction
 
x13
x26
DNA from Round A
137.6
TITANIUM Taq PCR buffer
16.0
208.0
416.0
dNTP mix (25mM each)
1.6
20.8
41.6
Primer B (100uM)
1.6
20.8
41.6
TITANIUM Taq
3.2
41.6
83.2
 
160
    


After mixing 160 uL PCR reaction, split into 8, 20 uL reactions
(multiple smaller volume reactions provide a higher yield)

Thermocycler program  
97°C
 3 min
25 cycles
 
97°C
 30 sec
40°C
 10 sec
50°C
 30 sec
72°C
 90 sec
72°C
 5 min


Step 2: Clean up PCR reactions
Pool 8 replicate PCRs and clean up on 1 Qiagen PCR cleanup column (~15 ug yield)
Elute 2x with 50 uL EB (100 uL total), speed vac concentrate to desired volume, total yield should be ~15 ug

ITEM COMPANY CAT. NO.
TITANIUM Taq Clontech 639209
Sequenase USB 70775Y