Flow Scheme:

(1) template preparations

(2) cycle-sequencing rxns

(3) product purification & preparations for the CEQ 8000

(4) sequencing runs in the CEQ

(5) data export to lab network I:/drives

(6) bioinformatic analyses of collected data

Networked Labs:
Lynn Bedard
Sharon Crary
Chet Fornari
Dan Gurnon
Wade Hazel
Jackie Roberts
H. Schneider
Janet Vaglia

Other Useful
Sites:

 

 

Actual cycle-sequencing reactions (for row 6 of the sequencing micro-titer plate) are shown below in our standard sequencing data sheet, which is designed according to the rows and columns of a standard micro-titer plate (one set-up sheet per row of the plate). We normally include a standard pUC18 reaction in every run for trouble-shooting any problems. You may view or download a blank form here; the sheet is adaptable to other primers by inserting more rows in that section. We plan to present all our methods in the near future on this page, according to the entries in the Flow Scheme (see side-bar).

DePauw University Biology Department
DNA Sequencing & Bioinformatics Lab 219 Olin: Sample Cycle Sequencing Data Sheet

NAME:
P&C

DATE:
3_28_06

EXPT.: BIO415 Pre-lab for MC1R RSAs

 ROW: 6-orange tubes labeled A-H (cross reference in your notebook)

Template name - conc. ng/ul

 

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

notes:

pUC18 - ( _ ng/ul) (NO heat treatment)

1

0.5

 

 

 

 

 

 

 

 

pUC18; (NO heat treatment)

2

 

0.5

 

 

 

 

 

 

 

1CFRh;

3

 

 

1

 

 

 

 

 

‘h’= high conc.

2CFHh;

4

 

 

 

  1

 

 

 

 

 

3EYgenh;

5

 

 

 

 

1

 

 

 

 

4EY294h;

6

 

 

 

 

 

1

 

 

 

5EPgenh;

7

 

 

 

 

 

 

1

 

 

6EP3h;

8

 

 

 

 

 

 

 

1

 

Sterile H2O to bring final plasmid template volume to 9 ul
before “Nick” program

 

6.5

8.5

8

8

8

8

8

8

no heat-nicking needed for std pUC18 or PCR

-47 Beckman primer 3.2 pmole/rxn final

 

2

 

 

 

 

 

 

 

 

-prm: use at 4 pmole/rxn final: M13 reverse - Invitrogen

 

 

0.2

0.2

0.2

0.2

0.2

0.2

0.2

 

Master Mix (Beckman-Coulter DTCS Quick Start)*

 

3*

3*

3*

3*

3*

3*

3*

3*

 

5X Sequencing Buffer (magic!)

 

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

 

1.3

1.3

1.3

1.3

1.3

1.3

1.3

1.3

 

final vol: 9 + 0.2 + 3 + 1.5 + 1.3  =
(for reactions C to H)

 

15

15

15

15

15

15

15

15

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*We sometimes use (as in this particular set-up) less than a "full" reaction, which is 8 ul of the DTCS Quick Start Master Mix in a 20 ul final reaction volume as recommended by Beckman-Coulter (so 0.4 ul DTCS in every ul of full reaction). As you can see here, we use only 3 ul of DTCS in a final reaction volume of 15 ul, which is equivalent to 0.2 ul of DTCS in a ul of reaction, and therefore a "half reaction" (0.2/0.4 = 0.5). Half reactions save some reagents, but accuracy of read-lengths may be less than for full reactions (e.g., 700 bp of quality sequence vs. perhaps 900 bp). See the Beckman-Coulter protocol for the typical, full reactions. And of course the absolute yields of our half-reactions in terms of nucleotide-bases synthesized would be 75% (or less depending on relative efficiencies) of BC's half-reactions (4 ul DTCS in a 20 ul final volume).

Remember the order of additions: (1) water, (2) template to make 9 ul (if template is a plasmid, heat “nick “ at this point and return to PCR cooler - pUC18 control plasmids supplied by Beckman-Coulter do not require the heat "nicking"). Next, (3) add primer, (4) Master Mix/SB and more water to make 6 ul; you can make a "mix" of these reagents, then add the 6 ul per reaction tube; e.g., a "mix" for 6 reactons would be 7.8 ul of water(from 6 x 1.3 ul water), 1.2 ul primer (from 6 x 0.2 ul), 18 ul of DTCS (from 6 x 3 ul), and 9.0 ul of SB (from 6 x 1.5 ul) to give a total volume all in one "mix" tube of 36 ul, which when divided by 6 (the number of tubes needing the final reagents) = 6.0 ul. So 9.0 ul of heat-nicked plasmid template prep + 6.0 ul of reagents = 15.0 ul. See the Beckman-Coulter protocol for the full 20 ul reaction and much simpler reaction set-up.

KEEP IN PCR COOLER AT ALL TIMES UNTIL COLLECTING VOLUME BY CENTRIFUGATION FOLLOWED BY PLACING SAMPLES IN THE THERMOCYCLER

NOTE: In spite of the appearance of this table, you will actually prepare a "mix" of (Master Mix + 5X SB + Primer + water) if you are using the same sequencing primer for several samples, as would be the case for plasmid vector templates, but not necessarity so for PCR fragments.

STOCK PRIMER CONCENTRATIONS for sequencing Plasmids:
Invitrogen M13 Forward (-20) = 20.35 uM/l = 20.35 pmole/ul
Invitrogen M13 Reverse = 19.25 uM/l = 19.25 pmole/ul
Invitrogen T7 =  16.4 uM/l = 16.4 pmole/ul

For help with these calculations, see: BioMath Calculators

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