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Klenow dsDNA synthesis

Back to protocols.

made on 5.07.2011

Materials

• 5’Comp Cy5 primer
  • 5’Cy5-GTC ATA CCG CCG GAGG
  • order from IDT at 1umole, HPLC purified
  • suspend to 500uM

• Library primers:
  • 5’ Linker - BINDING SITE – CCTCCGGCGGTATGAC
  • 5’ Linker generally: CCC

• Klenow Fragment 3’-5’ exo-
  • Order from NEB (Cat #: M0212L)

• dNTPs at 10mM each

Method

Synthesis reaction

3 μL	dNTP
3 μL	Buffer 2 (NEB Biolab)
2 μL	Library primer (150μM)
0.4 μL	5’Comp Cy5 primer
1 μL	Klenow 3’-5’ exo-
20.6 µL	dH2O
30 µL	Final volume

• Prepare a master mix without the library primer, and load 28μL into a 96 well plate.
• Add 2μL of Library primer to the 96 well plate.
• Cycle as follows:
  • 37°C for 1 hour
  • 75°C for 20 mins
  • ramp down to 30°C at 0.1°C/sec
  • hold at 4°C

• After the synthesis add 70μL of 0.5% BSA in dH2O to each well and transfer to a 384 well plate (flat bottom). Prepare dilutions as needed using 0.5% BSA dH2O.

• To set up the 384-well plate with 6 dilutions load 30uL 0.5% BSA dH2O into all columns except 1, 7, 13, 19.

• Transfer the column 1 of the klenow reaction (100uL total) to column 1 row A (A1) on the 384 well plate. Perform 5 dilutions of 70uL sample into the adjacent wells containing 30uL BSA dH2O. After the 5th dilution (in column 6) discard the remaining 70uL.

• Transfer column 2 of the klenow plate into A7 and perform dilutions.

• • Column3 -> A13, column 4 -> A19, Column 5->B1, col 6-> B7, col7->B13, col8 -> B19

• You should have 30uL of target DNA in each well of the 384 plate now. Add 30uL of 0.5% BSA dH2O to each well.

• The samples are now ready to be spotted.