End repair: Add 5-10 units of T4 DNApol and incubate at 37C for 5 minutes.
Make solution to 2.5 M NH4OAc and add 1 volume of 95% ethanol; let sit for 5 minutes at RT and spin at 14K x g for 20 minut....
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When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE, it is very important to determine how many extra nucleotides should be added to the 5’-e....
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Cleavage of PCR Products Directly After Amplification Reaction
In experiments with amplified DNA, restriction endonuclease (RE) digestion is usually performed directly in the PCR mixture, without tim....
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Place the sephadex measuring plate (MultiScreen ?Column Loader) on a clean piece of saran wrap. Pour some sephadex onto the plate (takes about 3.3 g). Scrape the surface with the Multiscreen metal pla....
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Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D. *Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, availa....
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PEG PRECIPITATION OF PCR PRODUCTS
This protocol can be used instead of EXO/SAP for removing excess primers and nucleotides from PCR products before cycle-sequencing. Protocol:1. After PCR, add the fo....
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Long PCR Reagents and Guidelines
(Modified from Cheng et al. (1))--------------------------------------------------------------------------------
General Guidelines for Long PCR Conditions and Enzym....
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Avoiding DNA Contamination in RT-PCR A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations. Although DNA contam....
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Gene-specific RT-PCR This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for t....
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Successful RT-PCR requires a high quality, intact RNA template. Use the following guidelines to help prepare this template: To minimize the activity of RNases that are released during cell lysis, incl....
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This procedure was first described by Bertrand et al (Proceedings of the National Academy of Science USA (1993) Vol. 90 pp. 3496-3500, and Nucleic Acids Research (1994) Vol. 22 pp. 293-300) to demonst....
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Reverse Transcriptase-PCR
This method was submitted by Jim Hutchins from the University of Mississippi Medical Center.
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