CGH of Biotin Labeled DNA vs Digoxigenin Labeled DNA
1) Reprecipitate DNA's
- Add the following DNA's to a 1.5 ml centrifuge tube, mixing with pipet:
20 ug Cot-1 DNA (~20ul)
~200 ng Biotin labeled DNA (~10ul)
~200 ng Digoxigenin labeled DNA (~10ul)
- Add 1/10th volume of 3M Na Acetate, mixing with pipet.
- Add 2.5 X (original) vol 100% EtOH to ppt DNA, vortex gently.
- Spin 30 mins at 14K rpm, 4 C.
- Decant supernatant; blot dry, being careful to avoid DNA pellet.
- Add 10 ul of MM1/H20 mix (70% MM1/30% H20).
- Carefully dissolve with pipet, and gently vortex.
- Quickly spin (1 sec) to bring volume to bottom of tube.
2) Denature slides:
- Select slides with spots in a good location, plenty of metaphases, and little or no cytoplasm around the metaphases or nuclei.
- Mark each spot on the back of the slide with a diamond pen.
- Prewarm slide on 37 C hot plate for 1 minute.
- Denature prewarmed slides in 70% formamide/2XSSC for 2.5-10 mins at 73 C inside a coplin jar (time will vary depending on slides).
- Denature no more than 3 slides at a time.
- Dehydrate slides through 70%, 85%, and 100% ethanols, 2 mins each.
- Wipe backs of slides and air dry upright on kimwipes.
- Place slides on 37 C warmer 1-2 minutes before adding probes.
- Denature probe mix at 70-75 C for 5 mins (optimum temp will vary with slides).
- Apply denatured probe immediatly onto warmed slide on the hot plate.
- Coverslip (18 mm) and seal with rubber cement.
- Let rubber cement dry 5 mins on warmer.
- Incubate for 2-3 days at 37 C in a humid chamber.
DAY 2: WASHES AND STAINING
- Remove rubber cement. Slide coverslips off gently.
- Wash 3X for 12 mins each at 45 C in 50% Formamide/2XSSC.
- Wash 1X for 10 mins at 45 C in 2X SSC.
- Wash 1X for 10 mins at RT in 2X SSC.
- Remove slide, quickly blot back (to remove excess liquid).
- Add 85 ul of 4X SSC/1% BSA preblock soln (kept in aliquots in freezer) and gently cover with a 24 X 50 mm coverslip.
- Incubate for 5 mins, then gently remove coverslip .
- Add 85 ul of a mix of anti-digoxigenin-rhodamine/FITC Avidin (at 1:200/1:400 in preblock solution; e.g. 2 ul anti-dig rhodamine + 1 ul FITC avidin in 397 ul of 4X SSC/1% BSA).
- Gently cover with a 24 X 50 mm coverslip.
- Incubate for 45-60 mins (protecting from light). Gently remove coverslip.
- Wash 10 mins each at RT in 4X SSC, then 4X SSC/0.1%Triton, then 4X SSC.
- Rinse slides 2X in ddH20 for 5-10 mins each, air dry upright.
- Apply 8.0 ul of 0.2 ug/ml DAPI in antifade (22 mm coverslip).
- Let sit at RT for at least 2 hours. Do not collect images the same day as Day 2 staining.
NOTES ON CGH:
Amplification of indirect signals:
If FITC-Avidin hybridization is very dim, but smooth, the signal can be amplified using our standard biotin amplification protocol. However, if it is dim and grainy, the signal will only get bright and grainy and the CGH profiles will be too noisy for interpretation.
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.
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 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.
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
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