Protocols - Aminoallyl (Indirect Incorporation) Labeling

 

  1. To 20ug total RNA add 2ul Random primers - Invitrogen
  2. Add RNase free water up to 18.5ul
  3. Mix well by flicking the tubes and incubate at 70C for 10 minutes to denature secondary structures.
  4. Snap freeze the RNA/primer mix in liquid Nitrogen.
  5. Centrifuge for 10 seconds at max rpm to collect the contents of the tube on the bottom.
  6. Add the following to each sample:
    a. 5X Superscript III Buffer – Invitrogen 6ul
    b. 0.2 M DTT - Invitrogen 3ul
    c. 50XAminoallyl-dNTP mix - Sigma 0.6ul
    d. Superscript II RT (200U/ul) - Invitrogen 2ul
  7. Mix by flicking the tube and microfuge quickly if necessary.
    Incubate at 42C for 3 hours to overnight.
  8. To end the RT reaction and hydrolyze the template RNA, add:
    1M NaOH 10ul
    0.5M EDTA 10ul
  9. Incubate at 65C for 15 minutes
  10. Add 10ul 1M HCl.

Clean-up of unincorporated nucleotides using Qiquick PCR kit

  1. Mix cDNA with 300ul PB and transfer to a QIAquick column in a 2ml collection tube.
  2. Microfuge for 1 minute at 14000 rpm to bind DNA to the column.
  3. Discard the flow-through and add 750ul Phosphate Wash Buffer.
  4. Spin at 14000 rpm for 1 minute.
  5. Repeat steps 3-4.
  6. Discard the flow-through and spin for an additional minute to dry the column.
  7. Transfer column to an eppendorf tube.
  8. Add 30ul 4mM KPO4 pH8.5
  9. Incubate for 1 minute and microfuge for 1 minute to elute.
  10. Repeat steps 7-8 for a total of 60ul.
  11. Your clean cDNA should now be in the eppendorf tube and the column can be thrown away.
  12. Dry the cDNA in the speed vac for 50-60 minutes.

Coupling

  1. Resuspend lyophilized cDNA in 4.5ul 0.1M Na2CO3 (to make dyes add 73ul DMSO to lyophilized dye, aliquot into 4.5ul, store at –80deg freezer)
  2. Add 4.5ul of the appropriate dye - Amersham to each sample.
  3. Incubate in the dark for 1 hour at room temperature.

Clean-up of Dyes using Qiagen’s Qiaquick PCR kit

  1. To the reaction add 35ul 100mM NaOAc
  2. Add 250ul PB, mix and pipet on to the QIAquick column.
  3. Microfuge for 30 to 60 seconds.
  4. Discard the flow-through and replace the column in the collection tube.
  5. Add 750ul PE to the column.
  6. Spin for 1 minute.
  7. Discard flow-though.
  8. Repeat steps 5-7.
  9. Spin the column for an additional minute.
  10. Place the column in a labeled eppendorf tube and discard the collection tube.
  11. Elute by adding 30ul EB directly to the column and incubate for 1 minute.
  12. Microfuge for 1 minute.
  13. Repeat steps 9-10
  14. Discard the column.
  15. (Optional: Remove 2ul of sample and mix with 2ul of loading buffer and run on a 1% agarose gel to confirm labeling.)
  16. Dry in the speed vac using the settings outlined above; this should take about 1 hour. After the samples are dry they can be used immediately or they should be covered with foil and used later that day.

Pre-Treatment of Slides

Preparation

  1. It is important to prepare everything you will need for the slide preparation before you start so that it will proceed quickly and smoothly.
  2. Fill a 100ml beaker with fresh MillliQ water and heat to 95C-100C.
  3. Make stock BSA: add 1g fractionV BSA to 84ml water and heat until dissolved, then add 15ml 20X SSC and filter. (make new every week)
    Make blocking buffer: mix 34ml 3X SSC with 3.8ml stock BSA(above) and 0.4ml 10% SDS in clean glass dish and place on shaker.
  4. Clean and fill a coplin jar with milliQ water.

Protocol

  1. Place the slide DNA side up in the Stratalinker in order to crosslink the DNA to the slide. Press energy and put in 2500 x 100 uJ.
  2. Place the slide DNA side up in the blocking buffer bath and turn the orbital shaker on low. Incubate for 2-10 minutes.
  3. Dip the slide in the coplin jar of MilliQ water 10-15 times to wash off the SDS.
  4. Place the slide in the 95C-100C beaker of MilliQ water and incubate for 1-2 minutes.
  5. Quickly place the slide in the prepared slide centrifuge and spin for 30-50sec. Use the slide immediately or store in a slide box desiccated at 2-8C.
  6. Place slide face up in the correct cartridge and put on the fluidics station. Slide is now ready to load.


Loading

  1. Resuspend lyophilized samples in 5ul of 10mM EDTA. (both cy3 and cy5 labeled samples will be resuspended in the same 5ul)
  2. Denature samples at 98deg. For 10 min.
  3. Aliquot 140ul of hyb buffer (hyb buffer #3 Ambion) and place in 68deg water bath until use.
  4. Mix hyb buffer with denatured sample and load onto slide.
  5. Hybridize slide for 16 hours.
    - slides are hybridized in the Genomic solutions GEN-TAC hyb station (Genomic Solutions)
    - slides are scanned with Applied Precision arrayWorx-E scanner
    - images are analyzed using softWorx tracker for spot finding

 

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