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Genisphere 3DNA Array 50 Kit Protocol

Genisphere 3DNA Array 50 Kit Protocol

Adapted from Genisphere Inc. 3DNA Array 50 Protocol

Bradfield Lab, October 2002


Step 1: Reverse Transcription and Cleanup


Non-Kit Materials Needed


Protocol

  1. Add appropriate amount of total RNA to PCR tube.

  2. Add 3ul RT primer to RNA (Vial 2-Make sure to add the right one cy3 or cy5)

  3. Add Rnase-Free water to bring volume to 20ul

  4. Mix gently with pipette

  5. Heat in thermocycler program “3DNA” (80/10min)

  6. Move tubes immediately into ice

  7. Add 1ul Superase-In Rnase inhibitor to each sample(Vial 4)

  8. Make up Master Mix


Master Mix (1 sample)

8ul 5x Superscript First Strand Buffer

2ul dNTP mix (Vial3)

4ul .1M DTT

2ul Superscript (add immediately before dispensing into samples)

3ul Rnase Free Water


  1. Mix Master Mix with pipette (do not vortex)

  2. Add 19ul of Master Mix to each PCR tube with RNA-primer mix

  3. Gently Mix with pipette and cycle on program “3DNA2” (42/2hours)

  4. Add 7.0ul of .5M NaOH/50mM EDTA

  5. Gently Mix and cycle on program “3DNA 3” (65/10min)

  6. Add 10ul 1M Tris-HCL, pH=7.5

  7. Combine probe sets to be compared (ie Treated cy3+Control Cy5) in a 1.5ml tube

  8. Add 16ul of TE to each probe set. The total volume of mixed probes should now be 130ul

  9. Thaw and Vortex Vial 5, linear acrylamide for 30 seconds

  10. Add 3ul of Vial 5 to samples

  11. Add 6ul 5M NaCL and mix

  12. Add 540ul of 100% EtOH, mix

  13. Incubate -30/30minutes

  14. Centrifuge max/ 20minutes at 4

  15. Aspirate supernatant

  16. Add 300ul of 70% EtOH

  17. Centrifuge max/ 5minutes at 4

  18. Aspirate supernatant carefully and dry completely in 37 degree incubator

  19. Add 10ul of nuclease free water (vial 10)

  20. Heat cDNA 65/10 minutes

  21. Vortex to resuspend

  22. Repeat last two steps until completely resuspended. Check resuspension by pipetting off water and checking for existence of pellet. Due to the linear acrylamide, the DNA may be harder to resuspend than normal.


Hybridization of cDNA for Overnight Incubation


Non-Kit Materials needed


Protocol


  1. Mix up pre-hyb buffer and heat to 45 in water bath (30mls/4 slides)

Pre-Hyb Buffer

  1. Add slides to hyb chamber and fill with pre-hyb buffer. Incubate at 45/45 minutes (proceed to step 4 during incubation). After 45 minutes, rinse slides by dipping into nano water three times and spin to dry in tabletop for 3minutes at 700rpm (place kimwipes in bottom of buckets). Place prepared chips in the hood and use within one hour.

  2. Prepare cover slips by dipping in .2% SDS, followed by water. Air dry in hood

  3. Thaw 2x formamide buffer (vial 7) and heat at 55/10minutes. Invert to resuspend

  4. Thaw cot-1 DNA and heat at 95/10minutes



  1. Add the following to each probe set:

    1. 17.5ul 2xbuffer (vial 7)

    2. 2ul Array dT Blocker (vial 9)

    3. 5.5ul Nuclease Free Water (vial 10)

    4. 1ul denatured Cot-1 DNA

  2. Heat probe mix for 10min at 77degrees in heated vortex

  3. Heat probe mix for 15min at 45 degrees in water bath

  4. Gently vortex cDNA probe mix

  5. Apply mix to pre-hybed slide, seal cover slip and place in slide holder

  6. Incubate at 45 in the dark overnight


Post cDNA Hybridization Washes


Materials


Protocol


  1. Wash slides for 10 minutes in 2XSSC, .2%SDS at 55 degrees. After 2minutes of wash cycle, check to make sure that cover slips have detached.

  2. Wash slides for 10 minutes in 2XSSC

  3. Wash slides for 10 minutes in .2X SSC

  4. Wash slides for 2 minutes in 95% EtOH

  5. Spin slides dry in tabletop at 700rpm/3minutes


3DNA Hybridization


***all following steps must be kept out of the light as much as possible


Materials


Protocol


  1. Thaw 2x formamide buffer (vial 7) and heat at 55/10minutes. Invert to resuspend.

  2. Thaw anti-fade reagent (Vial 8). Dilute anti-fade reagent 100x with 2x formamide buffer and mark with date. Anti-fade dilutions must be used within two weeks.

  3. Thaw both cy3 and cy5 capture reagents (Vials 1) at roomtemp in the dark for 20 minutes

  4. Heat the capture reagents at 55degrees for 10 minutes and vortex. Look for any particles that need to be resuspended ad repeat until all is in solution.



  1. Make Master Mix

Master Mix (1sample)

17.5ul 2X Formamide hyb buffer (Vial 7)

2.5ul 3DNA Capture Reagent Cy3 (Vial 1)

2.5ul 3DNA Capture Reagent Cy5 (Vial 1)

12.5ul Nuclease Free Water (Vial 10)

  1. Gently vortex Master Mix and heat 77/10min in heated vortex

  2. Heat Master Mix at 45/15min in water bath, spin briefly in centrifuge

  3. Prepare cover slips by dipping in .2% SDS, followed by water. Air dry in hood

  4. Add 35ul Capture Reagent Master Mix to slide and add coverslip

  5. Incubate 2 hours at 45 degrees in the dark


Final Wash of 3DNA hybridization


Materials


Protocol


1. Wash slides for 10 minutes in 2XSSC, .2%SDS at 55 degrees. After 2minutes of wash cycle, check to make sure that cover slips have detached.

2. Wash slides for 10 minutes in 2XSSC

  1. Wash slides for 10 minutes in .2X SSC

  2. Spin slides dry in tabletop at 700rpm/3minutes and place slides into a dark slide box.

  3. Scan on Avalanche