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WELCOME TO
The NTNU Plant Genetics Group
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INSTITUTE
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FACULTY
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N A R C
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Preparation of ddNTP master mixes, MIX 1
These master mixes are sufficient for approx. 75 sequencing samples (300 tubes, G A T C).
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ddG mix
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ddA mix
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ddT mix
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ddC mix
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80 µL dH2O
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80 µL dH2O
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80 µL dH2O
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80 µL dH2O
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22,5 µL 10 X Rx buffer*
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22,5 µL 10 X Rx buffer*
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22,5 µL 10 X Rx buffer*
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22,5 µL 10 X Rx buffer*
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100 µL "dNTP" mix**
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100 µL "dNTP" mix**
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100 µL "dNTP" mix**
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100 µL "dNTP" mix**
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25 µL 33P ddGTP
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25 µL 33P ddATP
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25 µL 33P ddTTP
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25 µL 33P ddCTP
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* 10 X reaction buffer: 260 mM Tris-Cl pH 9.5, 65 mM MgCl2
** dNTP mix: 7.5 µM of each dNTP. To avoid compressions use 7-deaza-dGTP in stead of ordinary dGTP.
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Transfer 3 µL to each corresponding tube labeled G, A, T, C.
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Overlay the mix with one drop of mineral oil, spinn down briefly and place
the tubes on ice.
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When the DNA/primer/enzyme mix is prepared (see next steps I or II),
place the tubes on the PCR block at 85° C.
I. Sequencing of agarase-treated PCR product
Short description of agarase treatment: Separate PCR products on a low melt agarose gel
with 1 X TAE as running buffer. Cut out the fragment with a scalpel and transfer the gel slice
to an eppendorf tube. Place the tube at 65° C for 5 min. (The gel slice will melt.)
Place it at 37° C for a couple of minutes. Add 1 µL of agarase (1 U/µL,
we use Sigma agarase), mix well and incubate at 37° C for 30-60 min.
| dH2O, Milli-Q |
H2O to final volume 10 µL. |
| 10 X Rx buffer |
1 µL |
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Agarase treated PCR product
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3-6 µL |
| Sequencing primer, 1 pmol/µL |
2 µL |
| Thermo sequenase 4 U/µL* |
1 µL |
* We use Amersham Thermo sequenase, catalog number US79750.
Keep the samples on ice until they are added to the ddNTP mix.
II. Sequencing of plasmid or lambda DNA, MIX 2
The concentration of plasmid and lambda DNA should not be lower than 50 ng/µL.
Normally, 1-2 µL from an ordinary miniprep is sufficient.
| dH2O, Milli-Q |
4.5 µL |
| 10 X Rx buffer |
1 µL |
| Plasmid or lambda DNA |
1.5 µL |
| Sequencing primer, 1 pmol/µL |
2 µL |
| Thermo sequenase (4 U/µL) |
1 µL |
* We use Amersham Thermo sequenase, catalog number: US79750.
Keep the samples on ice until they are added to the ddNTP mix.
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After you have aliquoted the ddNTP mixes into the labeled G, A, T and C
tubes and prepared the DNA/primer/enzyme mix (MIX 2), place the ddNTP mixes
on the PCR block at 85° C (hot start sequencing).
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Add 2.3 µL of MIX 2 to each tube labeled
G, A, T, C. Add the mix directly through the oil). Together with the ddNTP mix
(MIX 1), this should add up to a total volume of 5.3 µL.
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Start cycle sequencing. A typical cycle sequence file looks like this:
- 94° C for 1 min
- 50° C for 1 min
- 62° C for 1.5 min
- Stop the reaction with 5 µL stop solution.
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