Overview

First strand cDNA synthesis

1. Add 10 uL total RNA (20 ug) in DEPC H2O
   1 uL 100 pmol/ul T7-(T)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24)
2. Mix (quick spin if needed)
3. Heat @ 70 C, 10 min
4. Put in ice bucket
5. Add on ice to RNA/primer mix:
   • 4 uL 5X 1st Strand Buffer
     
   • 2 uL .1 M DTT
     
   • 1 uL 10 mM dNTPs
6. Heat @ 37, 2 min
7. Add 2 uL SSII RT (400 U total)
8. Mix (quick spin if needed)
9. Heat @ 42 C, 1 hour
10. Proceed to "Second strand cDNA synthesis"

Second strand cDNA synthesis

1. Ice all reagents and 1st strand tubes
2. Add to 1st strand tubes:
   • 91.33 uL DEPC H2O
   • 30 uL 5X 2nd Strand Buffer
   • 4 uL DNA POL I (40 Units)
   • 3 uL 10 mM dNTPs
   • 1 uL DNA Ligase (10 Units)
   • .67 uL RNase H (2 Units)
   
   
3. Mix (quick spin if needed)
4. Incubate @ 16°C, 2 hours
5. Store @ -80 C
   

Clean-up of dscDNA

1. Spin Phase-Lock tubes @ max, 30 sec
2. Add all of the cDNA reaction (approx. 150 uL)
3. Add equal volume buffer saturated phenol (or phenol/chloroform)
4. Vortex lightly
5. Spin @ max, 2 min
6. Transfer upper phase to new tube
7. Add
   • 1/2X volume 7.5 M NH4OAc (75 uL)
   • 2.5X volume 100% EtOH (375 uL)
   • 1 uL Glycogen (20 mg/mL)
   
   
8. Mix
9. Spin @ max, R.T., 20 min
10. Decant supernatant (watch for pellet)
11. Wash pellet twice with 80% EtOH
12. Speed vacuum to dry
13. Resuspend in 1.5 uL DEPC H2O

In Vitro Transcription

1. Thaw and room temperature all reagents
2. Make NTP mix (per tube):
   • 2 uL 75 mM ATP
   • 2 uL 75 mM GTP
   • 1.5 uL 75 mM CTP
   • 1.5 uL 75 mM UTP
   • 3.75 uL 10 mM Bio-11-CTP
   • 3.75 uL 10 mM Bio-16-UTP
   • 2 uL 10X Buffer
   
   
3. Add to cleaned dscDNA tube:
   • 16.5 uL NTP mix
   • 2 uL Enzyme mix (as provided in the kit)
   
   
4. Mix (quick spin if needed)
5. Incubate @ 37 C, 6 hours

IVT Clean-up

1. Add to IVT reaction tube:
   • 80 uL DEPC H2O
   • 350 uL RLT buffer
   
   
2. Mix
3. Add 250 uL 100% EtOH
4. Transfer sample to RNeasy spin column
5. Spin @ max, 15 sec
6. Transfer spin column to new collection tube
7. Add 500 uL RPE buffer
8. Spin @ max, 15 sec
9. Transfer spin column to new collection tube
10. Add 500 uL RPE buffer
11. Spin @ max, 2 min
12. Transfer spin column to new collection tube
13. Add 50 uL DEPC H2O to membrane of spin column
14. Let soak for 4 min
15. Spin @ max, 1 min
16. Repeat 13-15 using 1st elution as the 2nd elution
17. Take OD (1:50 dilution)
18. Run on a 1% agarose gel using denaturing sample buffer (See Appendix A)

Fragmentation of cRNA

1. Add to separate tube:
   • 40 ug cRNA (volume CANNOT exceed 64 uL)
   • X uL 5X Fragmentation Buffer

     Based on the volume of your cRNA, add the appropriate volume of 5X Fragmentation Buffer and adjust volume with DEPC H2O.
     For example,
     if you had 40 ug in 40 uL:
     40 uL cRNA (40 ug)
     10 uL 5X Fragmenation Buffer
     50 uL Total Volume
     

     or

     40 ug in 50 uL:
     50 uL cRNA (40 ug)
     13 uL 5X Fragmentation Buffer
     2 uL DEPC H2O
     65 uL Total Volume
     

2. Mix
3. Heat @ 95, 35 min
4. Add:
   • 450 uL 2X STT
   • 9 uL 10 mg/mL Herring Sperm DNA
   • 9 uL 948 Control Oligo or Control Oligo B2 (5'-Bio-GTCAAGATGCTACCGTTCA-3')
   • 9 uL 100X Bio B, C, D, and Cre
   • 0.5 mg/ml acetylated BSA
   
   
5. Adjust volume with DEPC H2O to 900 uL total volume
6. Store @ -80 C

• Hybridization onto Chips
• Fluidics
• Pre-Scanning: Creating .exp files
• Washing & Staining
• Scanning

Information about DNA Chips and Equipment

Appendix

1. Make Sample Buffer:

• .05 uL 10 mg/mL Ethidium Bromide
• .5 uL 10X MOPS
• 5 uL deionized-Formamide
• 1.75 uL 37% Formaldehyde
• 1 uL 10X Loading Dye
• 1.7 uL DEPC H2O

2. Add 10 uL Sample Buffer to each sample and controls to be run

3. Heat @ 65 C, 10 min

4. Run on 1% Agarose gel