Agilent Labeling

From Genome@Yale

Set the heat block to 95°C; thaw out primers, dNTP labeling mix, Cy5- and Cy3- dCTP.

BioPrime Array CGH protocol from Invitrogen for reference. Please note, volumes are slightly different.

1. Set up random priming reaction - components for the reaction are from Invitrogen CGH labeling kit

2ug of LM-PCR DNA - bring the volume to 40uL total with dH2O
35uL 2.5X random primer solution

2. Vortex 30 seconds

3. Place the tubes into the 95°C heat block for 5 minutes

4. Transfer the tubes immediately to aluminum block chilled on ice for 5 minutes

5. Make Labeing Mix

components               1x     4.5x
10X dNTP labeling mix    8.2uL  36.9uL
Cy5 or Cy3 dCTP          1.5uL  6.75uL
Klenow (40U/ul)          1.5uL  6.75uL
dH2O                     1.8uL  8.1uL
total                    13uL   

6. Spin down the denatured primer-DNA mix and add 13uL labeling mix

7. Mix thoroughly by pipetting

8. Incubate 3 hours at 37°C

9. Add 9uL stop buffer (from kit) to each tube and vortex

10. Clean up samples using Invitrogen CGH column

1. Add 0.4mL Purification Buffer A, vortex 30 seconds
2. Load the purification column
3. Spin the column at 8000g for 1 minute, discard flow through
4. Add 0.6mL Purification Buffer B
5. Spin the column at 8000g for 1 minute, discard flow through
6. Add 0.2mL Purification Buffer B
7. Spin the column at 8000g for 1 minute, discard flow through
8. Transfer the column to new amber tube
9. Add 50uL dH2O and incubate 1 minute at room temperature
10. Spin the column at 8000g for 1 minute
11. OD labeled DNA using Nanodrop