Preparation of DNA & RNA Probes

 

Protocol 1. Standard DNA random primers labeling protocol.

 

1. Denature 25-100ng DNA dissolved in 20ul of ddH2O in a microcentrifuge tube by heating for 5 minutes in a boiling water bath, then immediately cool on ice.

 

2. Add the following reagents on ice and mixt gently.

 

1 ul 500uM dATP

1 ul 500uM dGTP

1 ul 500uM dTTP

10ul 5x random primers solution

5 ul (about 50 uCi) alpha-32P-dCTP

ddH2O to a total volume of 49 ul.

 

3. Add 1 ul Klenow enzyme. Mix gently but thoroughly. Centrifuge 305 seconds.

 

4. Incubate at 37 centigrade for 10-15 minutes.

 

5. Add 5 ul stop buffer (0.5 M EDTA).

 

6. This labeled DNA fragments can be used for hybridization.

 

 

Protocol 2. Synthesis of single strand DNA probe

 

Protocol 3. Radiolabeling probes by PCR

 

Protocol 4. Synthesis of cDNA probes from mRNA 

 
 

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Ch 1.General Lab Techniques

Ch 2.Molecular Separation

Ch 3.DNA and RNA

Ch 4.Genetics

Ch 5.PCR Serials

Ch 6.Protein

Ch 7.DNA Protein Interactions

Ch 8.Immunohistoch / immunology

Ch 9.Cellular Biology

Ch 10.GC/MS, NMR and Proteomics

Ch 11.Animal Experiments

Ch 12.Worm: C. Elegans

Ch 13.HPLC and TLC

Ch 14.Buffers formats in Lab.

Ch 15.Other Resources

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