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CGH of DIRECT LABELED DNA

CGH of DIRECT LABELED TEST DNA vs NORMAL DNA

This CGH Protocol is used for DNA of good quality when available in sufficient amounts. We usually do replicate hybridizations using samples labeled "inversely" (reversing the label for test and normal DNA's). If appreciable artifact occurs, then alternative labels are tried.

DAY 1:

1) Reprecipitate DNA's

2) Denature slides:

3) Hybridization:

DAY 2: WASHES AND STAINING

NOTES ON CGH VARIABLES:

Cot-1 DNA:

Cot DNA varies from lot to lot. We have requested that a single Lot be put on reserve for our use. We then measure its concentration (1 mg/ml) and test it for CGH.

Probe DNA:

The amount of probe DNA used can vary, and should be adjusted depending on the intensity of the product. We generally use the following voumes of the ~1 ug/50 ul nick translation product:

Fresh DNA: 12 ul
Paraffin DNA: 45 ul
PCR product from fresh dna: 20 ul
PCR product from microdissected DNA: 45 ul

Probe size

Probe size is probably the most important aspect of CGH success. For good quality DNA (from fresh tissue), size should be adjusted by repeating the nick translation. It is best to use probes which are a smear between 0.3 - 2.3 kb. Size can be adjusted by reducing or increasing the standard time for nick translation from 60 mins (using 2.5-5 ul of DNAse/Pol-1 enzyme mix. Paraffin samples also should be this size, although often they appear bigger. PCR products are usually smaller, ranging from 0.1 - 1.5 kb.

Slides:

The quality of the slides used is the second major variable in CGH. Each new batch should be tested under different conditions, such as adjusting denaturation time and temperature.

Slide storage time

Storage time is another variable which affects the CGH quality. After some time they may give variable results. This is why it is important to run a control sample. Usually if the chromosome banding is very good, the cgh may suffer. You may need to sacrifice the banding slightly in order to get optimum cgh.

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S. DeVries / F. Waldman

Email questions to Sandy DeVries at the UCSF Cancer Center (devries@cc.ucsf.edu)

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