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Title: semi quantitative RT-PCR Post by: Felicis on Nov 27, 2014 Hello, I have a few questions on Semiquantitative RT-PCR
1) what do you expect to see on a gel containing your target and housekeeping genes if the results are positive? i.e. how many bands and changes in intensity etc 2) Why should both target and housekeeping gene be amplified in the same tube? Is it something to do with the same conditions are needed for both genes? 3) What are the ingredients for RT-PCR and what are their roles? 4) Someone suggested VEGF with primers and correct conditions can produce 3 bands on a gel, along with the housekeeping gene, why is this? Thanks in advance, I have tried searching on the internet but most results are vague or explain real time pcr Title: Re: semi quantitative RT-PCR Post by: habiba on Nov 27, 2014 2) Why should both target and housekeeping gene be amplified in the same tube? Is it something to do with the same conditions are needed for both genes? When RNA is the template, performing amplification in the same tube provides some normalization against variables such as RNA integrity and reverse transcription efficiencies. 3) What are the ingredients for RT-PCR and what are their roles? - Template DNA 50-100ng/μl - Reaction buffer (Tris-HCl, ammonium ions, KCl), magnesium ions, bovine serum albumin) This buffer provides the ionic strength and buffering capacity needed during the reaction. - MgCl2 - 1.5- 3mM - dNTPs -Equimolar ratios, 200 μM each dNTP - Primers (0.1 and 0.5 μM) - DNA polymerase -1-2 unit/25 μl reaction |