[an error occurred while processing this directive] I. PREPARATION OF EMBRYOS

PCR Protocol for Genomic DNA

Regent

Single rxn

1plate

2plates

3plates

4plates

6plates

8plates

10XcDNA PCR Buffer

3.0

330

660

990

1320

1980

2640

100mM dATP

0.06

6.6

13.2

19.8

26.4

39.6

52.6

100mM dCTP

0.06

6.6

13.2

19.8

26.4

39.6

52.6

100mM dGTP

0.06

6.6

13.2

19.8

26.4

39.6

52.6

100mM dTTP

0.06

6.6

13.2

19.8

26.4

39.6

52.6

20uM 5'primer

3

xxxxx

xxxxx

xxxxx

xxxxx

xxxxx

xxxxx

20uM 3'primer

3

xxxxx

xxxxx

xxxxx

xxxxx

xxxxx

xxxxx

DEPC H2O

18.66

2052.6

4105.2

6157.8

8210.4

12315.6

16420.8

DyNAzyme PCR Poly

0.6

66

132

198

264

396

528

Genomic DNA (50ng/ml)

1.5

165

330

495

660

990

1320

TOTAL

30

2640

5280

7920

10560

15840

21120

  1. Mix all reagents thoroughly (Multi tube Vortexer)
  2. Add 24 ul of the mix into each well using a mulichannel pipette (Brand Transferpette-12)
  3. Add 3 ul of individual 5' primer into each well
  4. Add 3 ul of individual 3' primer into each well
  5. Mix thoroughly (use Multi-tube Vortexer) and spin the plate down (Eppendorf Centrifuge 5810R) ,
  6. Place plate in PCR machine (GeneAmp PCR System 9700)
  7. Use cycling conditions below
  8. Run Gel
  9. The samples are now ready for G50 purification

95C 7min 30sec

32 Cycles

94C 30sec

54C 45sec

72C 1min

72C 10min

4C .........

Product

Supplier

Catalog#

Size/V/m

Deoxynucleotides

-dATP

-dCTP

-dGTP

-dTTP

Promega

Promega

Promega

Promega

U120A

U122A

U121A

U123A

400 ul

400 ul

400 ul

400 ul

Individual Primers

DyNAzyme EXT DNA Polymerase

with 10x PCR Buffer

MJ Research

F-505L

500 ul