NSF Soybean Functional Genomics Project
Vodkin Laboratory, University of Illinois |
Preparation of DNA for Microarrays,
page 2, 3
Steve Clough/Reena Philip
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PCR
Data/Reaction Sheet
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Purpose: Prepare
PCR products for microarraying by amplification of diluted plasmid
DNAs from 96-well plates with Gibco/BRL Taq polymerase using MJ
DNA Engine.
Sample Plate: _______________________________________________
Rxn Date / User initials: ________________________________________
MJ Plate type: polypropylene: 200 _l Microseal Skirted (MSP-9621)
Vector: ______pSPORT
Other:____________________
Primers: M13 Universal Forward (-21) and M13 Reverse (-24)
(5' GTA AAA CGA CGG CCA GT 3')
(5' AAC AGC TAT GAC CAT G 3')
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| REACTION |
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Mix the following together as a master mix in a sterile trough
and use electronic multichannel pipettor to aliquot 48 ml in each
well of 96-well V-well polypropylene plate (if not using a plate
from MJ Research, verify that it fits well with the DNA Engine).
| GIBCO / BRL |
Total volume: 50 ml
Enzyme |
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Lot # ________________ |
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100 rxn master mix
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1 rxn |
[final] |
1 plate |
2 plates |
4 plates |
| sdH2O |
30.5 ml |
--- |
3.05 ml |
6.10 ml |
12.2 ml |
| 10X PCR Buffer |
5 ml |
1 X |
500 ml |
1 ml |
2 ml |
| 50 mM MgCl2 |
2 ml |
2 mM |
200 ml |
400 ml |
800 ml |
| 10 mM dNTPs (2.5 mM each) |
5 ml |
0.25 mM |
500 ml |
1 ml |
2 ml |
| 20 mM Universal Primer |
2.5 ml |
1 mM |
250 ml |
500 ml |
1 ml |
| 20 mM Reverse Primer |
2.5 ml |
1 mM |
250 ml |
500 ml |
1 ml |
| Taq Polymerase (5 U/ml) |
0.5 ml |
0.05 U/ml |
50 ml |
100 ml |
200 ml |
| (catalog no: 18038-042) |
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Add 2 ml of the diluted cDNA
(~0.5 ng/ml) to each well using
Finnpette manual multichannel pipettor. Swirl gently with tips to
mix. Mark above each column of wells after adding DNA to indicate
that DNA was added. Cover plates very well with foil tape. Spin
at 4000 rpm for 1min at RT.and place into MJ PTC-200 DNA Engine.
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PCR PROGRAM |
| Name: Gibco 39 |
1. 94c 0:01:00 |
Heat: calculated |
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2. 92c 0:00:30 |
Vessel type: tube (even for |
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3. 56c 0:00:45 |
plates. Tubes=polypropylene). |
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4. 72c 0:03:30 |
Volume: 50 ml |
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5. GoTo step 2, 39 times |
Lid heated?: Yes |
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6. 72c 0:05:00 |
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7. 4c 0:00:00 |
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8. END |
Run time = 4 hr |
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Preparation
of 10 mM dNTPs for PCR
Make 10 mM stock concentrations of each nucleotide by adding
the following amounts of sterile nanopure water to each 10 mg vial
(do not try to weigh the powder):
| dATP |
Sigma D-6500 |
FW 535.1 |
Dissolve 10 mg in 1.87 ml H2O |
| dCTP |
Sigma D-4635 |
FW 511.1 |
Dissolve 10 mg in 1.96 ml H2O |
| dGTP |
Sigma D-4010 |
FW 507.2 |
Dissolve 10 mg in 1.97 ml H2O |
| dTTP |
Sigma T-0251 |
FW 482.2 |
Dissolve 10 mg in 2.07 ml H2O |
Add equal volumes of the 10 mM stocks and the final concentration
of each nucleotide will be 2.5 mM (each nucleotide is thus diluted
4 times in the final volume and has a concentration of 2.5 mM) and
the concentration of nucleotides in general (dNTPs) will remain
10 mM. Since dATP has lowest volume (1.87 ml), add 1.75 ml
of each into a sterile 15-ml orange cap tube. Final volume equals
7.0 ml. Distribute 1-ml aliquots into 14 microfuge tubes and store
at -70°C. Store the extra individual nucleotide 10 mM stocks at
-70°C.
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Preparation of 20 mM Primers:
Purchase 25 nmoles from the Keck Center Sequencing Lab. Dissolve in
1.25 ml sterile nanopure water (25nmoles/1.25 ml = 20 nmole/ml or
20 mmole/L which is 20 mMolar). Let set on desk for several minutes
to ensure primers are well dissolved. Store at -20°C. (Use 1 ml of
primer for four plates of PCR. Store the remaining 250 µl at -20°C).
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