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vincio

Member Since 20 Apr 2009
Offline Last Active Apr 29 2013 09:46 PM
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Topics I've Started

pcr 10X buffer preparation

28 April 2013 - 03:42 AM

Hello,
I am tryng to prepare home made 10x buffer (750mM tris ph 9, 500mM KCL and 200mM NH4SO4, 1% tritonX). My peparation is not working and I have two doubts to resolve:

1) I am using Tris-HCL solution 2M ph 9 made from tris-hcl salt bring to ph with NaOH : is it this salt correct for PCR? Have I to use Tris-base ? I am thinking in some negative iteration of Na+ ions..

2)I use ph 9 tris but in final solution the ph is 8.0-8.2 I have to further adjust ph ?

thank you for any help

Vincenzo

digestion and labelling of large ammount of DNA

21 July 2012 - 03:38 AM

Hello ,I want to apply this protocol to produce fluorescent labelled restriction fragment:

1. Digest 50µg of PhiX174 DNA with DdeI and CfoI endonuclease in 200µl reaction).

2. Following the restriction digest, add
    80 µl dATP, dCTP, dGTP, (2.5 mM each)
    8 µl of 1 mM 12-Texas Red dUTP (Molecular Probes), or any other dUTP or dTTP with a fluorescent label
    8 µl Klenow Fragment of DNA Polymerase (40 units)


my question is: can be the concentration of reagent be optimezed?
For example is necessary to perform the digestion in 200ul of reaction?How many ul reaction are needed to digest 50ug of phage dna?

Thank you for any help
Vincenzo

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