DePauw University Biology Department

DNA Sequencing & Bioinformatics Lab 219 Olin: Cycle Sequencing Data Sheet

 

NAME:

 

DATE:

 

EXPT.:

 

 ROW:

 

Template name-conc. (ng/ul)

 

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

notes:

 

1

 

 

 

 

 

 

 

 

 

 

2

 

 

 

 

 

 

 

 

 

 

3

 

 

 

 

 

 

 

 

 

 

4

 

 

 

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

6

 

 

 

 

 

 

 

 

 

 

7

 

 

 

 

 

 

 

 

 

 

8

 

 

 

 

 

 

 

 

 

Sterile H2O to bring final plasmid template volume to 9 ul

before “Nick” program

 

 

 

 

 

 

 

 

 

 

*primer:

 

 

 

 

 

 

 

 

 

 

*primer:

 

 

 

 

 

 

 

 

 

 

Master Mix

 

3

3

3

3

3

3

3

3

 

5X S.B. (magic buffer)

 

1.5

1.5

1.5

1.5

1.5

1.5

1.5

1.5

 

Sterile H2O to bring mix vol. + primer to 6 ul

 

 

 

 

 

 

 

 

 

 

vol: 9 + x ul + 3 + 1.5 + y ul  =

 

15

15

15

15

15

15

15

15

final volumes

x ul = primer volume

 

 

 

 

 

 

 

 

 

 

y ul = 1.5 ul – x ul primer vol

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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 (for help, see BioMath Calculators)