10. Transfer slides to a clean, dust free slide box and let stand overnight before hybridizing.
2.10. PRINTING
The variety of printers and pens for transferring PCR products from titer plates to slides precludes highly detailed descriptions of the process. The following steps provide a general description of the processing.
1. Pre-clean the print pens according to the manufacturer's specification.
2. Load the printer slide deck with poly-L-lysine coated slides (Supplemental Protocol 3).
3. Thaw the plates containing the purified EST PCR products and centrifuge briefly, two minutes, at 1000 rpm in a horizontal microtiter plate rotor to remove condensation and droplets from the seals before opening.
4. Transfer 5 to 10 µl of the purified EST PCR products to a plate that will serve as the source of solution for the printer.
Printing with quill-type pens usually requires that the volume of fluid in the print source is sufficiently low, that when the pen is lowered to the bottom of the well, it is submerged in the solution to a depth of less than a millimeter. This keeps the pen from carrying a large amount of fluid on the outside of the pen shaft and producing variable, large spots on the first few slides printed.
5. Run a repetitive test print on the first slide. In this operation, the pens are loaded with the DNA solution, and then the pens serially deposit this solution on the first slide in the spotting pattern specified for the print.
This test is run to check the size and shape of the specified spotting pattern, and its placement on the slide. It also serves to verify that the pens are loading and spotting, and that a single loading will produce as many spots as are required to deliver material to every slide in the printer.
6. If one or more of the pens is not performing at the desired level, re-clean or substitute another pen and test again.
7. If all pens are performing, carry out the full print.
RNA EXTRACTION
This protocol details the methods used to extract RNA from cells, purify the RNA by a combination of phase extraction and chromatography, and prepare a labeled cDNA copy of the message fraction of the purified RNA. The protocol also describes the process of making fluorescent cDNA representations of the message pools within the isolated total RNA pools. This is accomplished by using the pure total RNA as a substrate for reverse transcription in the presence of nucleotides derivatized with either a Cy3 or a Cy5 fluorescent tag.
Materials
Trizol Reagent (#15596-018, Life Technologies, Rockville, MD)
RNeasy Maxi Kit (# 75162, Qiagen, Valencia, CA)
Chloroform
Ethanol (200 Proof USP Ethyl Alcohol)
DPBS (Dulbecco's phosphate buffered saline)
3M sodium acetate (pH 5.2)
dATP, dCTP, dGTP, dTTP, 100 mM each, store frozen, -20°C (#27-2035-02, Pharmacia, Peapack, NJ)
pd(T)12-18 resuspend at 1mg/ml, and store frozen -20°C ( #27-7858, Amersham Pharmacia Biotech)
Anchored oligo primer (anchored;5'-TTT TTT TTT TTT TTT TTT TTV N-3')
resuspend at 2mg/ml, store frozen -20°C (e.g. # 3597-006, Genosys)
CyTM3-dUTP, 1 mM, and CyTM5-dUTP, 1 mM, store -20°C, light sensitive
RNasinâ Rnase inhibitor, store -20°C (#N211A, Promega)
SUPERSCRIPTTM II Rnase H` Reverse Transcriptase Kit, store -20°C, (#18064-014, Life Technologies, Rockville, MD)
C0t-1 DNA, 1mg/ml, store frozen -20°C (#15279-011, Life Technologies, Rockville, MD)
0.5M EDTA( pH 8.0)
1 N NaOH
1M TRIS-HCL (pH7.5)
TE pH 7.4
DEPC water 50 X Tris Acetate Buffer
15 ml round bottom polypropylene centrifuge tubes
50 ml conical polypropylene centrifuge tubes
1.5 ml Eppendorf tubes
0.2 ml thinwall PCR tube
MicroCon 100 ( Amicon Cat No. 42412)
High speed centrifuge for 15 ml tubes
Clinical centrifuge with horizontal rotor for 50 ml conical tubes
Tissue homogenizer (e.g. Polytron PT1200 with Polytron-Aggregate-Dispergier-und-Mischtechnik 147a Ch6014 #027-30-520-0, Brinkmann Instruments Inc., Westbury, NY)
Reagents and Solutions
RPE Buffer
Add 4 volumes of ethanol per volume of RPE concentrate supplied in Quiagen Kit.
RW1 Buffer
Supplied in Qiagen Kit
75% EtOH
Ethanol (100%) 375 ml
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