DePauw University Biology Department
DNA Sequencing & Bioinformatics Lab
219 Olin: Cycle Sequencing Data Sheet
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EXPT.: ROW: |
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Template name-conc. (ng/ul) |
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(A) |
(B) |
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(E) |
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(G) |
(H) |
notes: |
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2 |
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8 |
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Sterile H2O
to bring final plasmid template volume to 9 ul before
“Nick” program |
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*primer: |
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*primer: |
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Master Mix |
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3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
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5X S.B. (magic buffer) |
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1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
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Sterile H2O
to bring mix vol. + primer to 6 ul |
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vol: 9 + x ul + 3 + 1.5 + y ul = |
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15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
final volumes |
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x ul = primer volume |
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y ul = 1.5 ul
– x ul primer vol |
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Remember the order of additions: (1) water, (2) template (if template
is a plasmid, heat “Nick” at this point and return to PCR cooler: then add (3)
primer, (4) Master Mix/SB:
9 ul plasmid/water
“nicked” template + 6 ul reaction mix = 15 ul total rxn
volume
KEEP IN PCR COOLER AT
ALL TIMES UNTIL COLLECTING VOLUME BY CENTRIFUGATION FOLLOWED BY PLACING SAMPLES
IN THE THERMOCYCLER
NOTE: As shown in the
table, you will prepare a solution of (Master Mix + 5X SB +Primer) if you are
using the same sequencing primer for several samples
*STOCK PRIMER
CONCENTRATIONS for sequencing Plasmids:
-47 Beckman primer at
3.2 pmole/rxn; normally use 2 ul per reaction
Invitrogen
M13 Forward (-20) = 20.35 uM/l = 20.35 pmole/ul
Invitrogen M13 Reverse = 19.25 uM/l = 19.25 pmole/ul; use 0.21
ul per reaction
Invitrogen T7 = 16.4 uM/l = 16.4 pmole/ul