Download Protocol Purifying DNA for Sequencing ***Change pipet tips EVERY

Protocol
Purifying DNA for Sequencing
***Change pipet tips EVERY time you pipet something***
Summary: We will be sequencing the DNA sequences of the three virus genes you amplified
using pcr, but first, we need to clean up the DNA. Pcr reactions contain DNA, primers, buffer,
MgCl2, taq polymerase, and dNTPs. We must remove everything except for the DNA to get it
sequenced.
1) Label 3 microtubes tubes with 500 µl-capacity:
psbA
(virus #)
DNApol
(virus #)
g23
(virus #)
2) Add 200 µl of Buffer PB to each tube (you can use the same pipet tip):
200 µl
200 µl
Buffer
PB
200
µl
psbA virus
DNApol virus
g23 virus
3) Get your pcr products from your viruses (you do NOT need your positive or negative control)
4) Add 40 µl of your pcr products to the appropriate tubes with the buffer
**this will use ALL of your pcr product**
psbA pcr product
DNApol pcr product
g23 pcr product
40 µl
40 µl
40 µl
psbA virus
DNApol virus
g23 virus
5) Label 3 microtubes tubes with 2 ml-capacity; **label ON THE SIDE of the tube**
psbA
(virus #)
DNApol
(virus #)
g23
(virus #)
6) Put MinElute columns into the 3 labeled microtubes with 2 ml-capacity:
psbA
(virus #)
DNApol
(virus #)
g23
(virus #)
7) Pipet all of your samples mixed with Buffer PB into the MinElute columns:
psbA virus
DNApol virus
g23 virus
240 µl
psbA virus
240 µl
DNApol virus
240 µl
g23 virus
8) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute.
9) Pour out the liquid that went through the column into a waste beaker.
-KEEP the column and the tube
10) Add 750 µl of Buffer PE to each MinElute column:
psbA virus
µl
750
750 µl
Buffer
PE
75
DNApol virus
0µ
l
g23 virus
11) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute.
12) Pour out the liquid that went through the column into a waste beaker.
-KEEP the column and the tube
13) Label 3 microtubes tubes with 1.5 ml-capacity; **label ON THE SIDE of the tube**
psbA
(virus #)
DNApol
(virus #)
g23
(virus #)
14) Transfer your MinElute columns into the newly-labeled microtubes with 1.5 ml-capacity
15) Add 10 µl of Buffer EB to each MinElute column
- put the buffer DIRECTLY onto the column membrane:
psbA virus
10
µl
10 µl
Buffer
EB
10
DNApol virus
µl
g23 virus
16) Let the columns sit for 1 minute.
17) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute.
18) KEEP THE LIQUID THAT WENT THROUGH THE MINELUTE COLUMN
-This is your DNA!!!
19) Discard the MinElute Columns and close your tubes.
19) Put your recovered DNA into the ice bucket.