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INFORMATION FOR

Mapping

Protocol for PCR and Restriction Digest for SNP Detection

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1. Dilute primers to proper concentration (100mM) with 1/10X TE
2. Mix forward and reverse primers 1:1 (ie 25ul:25 ul)
3. Prepare PCR mix for 50 ul reactions
a. Use PCR mix calculator available on our website
4. Aliquot 48 ul PCR mix to each well
5. Aliquot 1 ul appropriate DNA to wells
6. Aliquot 1 ul appropriate primer mix to wells
7. Spin plate down briefly
8. Run PCR using Khokha Lab “Frog 58S” program

a. PCR steps:

i. 94 for 2 min
ii. 94 for 10 sec
iii. 58 for 30 sec
iv. 72 for 30 sec
v. Go to 2, 39 times
vi. 72 for 5 min
vii. 15 forever

9. Check for DNA on regular 2% agarose gel

a. 10ul DNA+1ul Loading dye
b. Note: Large gel tray uses 250 ml agarose

10. Prepare digest

a. Organize primers, restriction enzymes and appropriate buffers using template well table to avoid confusion later
b. Quantitate amount of each buffer needed and prepare

i. Each enzyme uses either Buffer 2 or 3; prepare buffers using protocol from the Aroian Lab at UCSD
ii. Use SNP digest buffer calculator available on our website

c. Add 7 ul appropriate buffer to each well
d. Add 8 ul appropriate DNA to each well
e. Add 0.5 ul appropriate enzyme to each well, or add enzyme to mix if only 1 enzyme

11. Spin plate down briefly
12. Digest at 37C for 4 hours if using PCR machine. We find Overnight is best otherwise (incubator)

a. Note: Khokha PCR machine has 37C program.

13. Run out 10 ul uncut DNA alongside entire volume corresponding digest on 4% 50:50 regular agar: NuSeive GTG agarose gel. See protocol:Preparing 4% 50:50 Regular:NuSeive Gel

a. Use 250 ul agarose for large tray. b. Run 120v for 45 minutes to 1 hour, with 100 bp ladder.
c. Note: Need to raise two end combs up using bits of lab tape to prevent holes forming in bottoms of wells.