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{\rtf1\mac\ansicpg10000\uc1 \deff0\deflang1033\deflangfe1033{\upr{\fonttbl{\f4\fnil\fcharset256\fprq2{\*\panose 02000500000000000000}Times;}{\f7\fnil\fcharset256\fprq2{\*\panose 020b0503030404040204}Geneva;} }{\*\ud{\fonttbl{\f4\fnil\fcharset256\fprq2{\*\panose 02000500000000000000}Times;}{\f7\fnil\fcharset256\fprq2{\*\panose 020b0503030404040204}Geneva;}}}}{\colortbl;\red0\green0\blue0;\red0\green0\blue255;\red0\green255\blue255;\red0\green255\blue0; \red255\green0\blue255;\red255\green0\blue0;\red255\green255\blue0;\red255\green255\blue255;\red0\green0\blue128;\red0\green128\blue128;\red0\green128\blue0;\red128\green0\blue128;\red128\green0\blue0;\red128\green128\blue0;\red128\green128\blue128; \red192\green192\blue192;}{\stylesheet{\widctlpar\adjustright \loch\af4\hich\af4\dbch\f4\cgrid \snext0 Normal;}{\*\cs10 \additive Default Paragraph Font;}}{\info{\title PEG Precipitation of PCR products}{\author Travis Glenn}{\operator Travis Glenn} {\creatim\yr2000\mo10\dy2\hr14\min31}{\revtim\yr2000\mo10\dy2\hr14\min35}{\version1}{\edmins4}{\nofpages2}{\nofwords427}{\nofchars2439}{\*\company SREL}{\nofcharsws2995}{\vern99}}\margl1440\margr1440 \widowctrl\ftnbj\aenddoc\formshade\viewkind1\viewscale100\pgbrdrhead\pgbrdrfoot \fet0\sectd \linex-32767\endnhere\sectdefaultcl {\*\pnseclvl1\pnucrm\pnstart1\pnindent720\pnhang{\pntxta .}}{\*\pnseclvl2\pnucltr\pnstart1\pnindent720\pnhang{\pntxta .}} {\*\pnseclvl3\pndec\pnstart1\pnindent720\pnhang{\pntxta .}}{\*\pnseclvl4\pnlcltr\pnstart1\pnindent720\pnhang{\pntxta )}}{\*\pnseclvl5\pndec\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl6\pnlcltr\pnstart1\pnindent720\pnhang{\pntxtb (} {\pntxta )}}{\*\pnseclvl7\pnlcrm\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl8\pnlcltr\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}{\*\pnseclvl9\pnlcrm\pnstart1\pnindent720\pnhang{\pntxtb (}{\pntxta )}}\pard\plain \qc\widctlpar\adjustright \loch\af4\hich\af4\dbch\f4\cgrid {\b\fs32 \hich\af4\dbch\af4\loch\f4 PEG Precipitation of PCR products}{\b\fs32 \par }\pard \fi720\sl-360\slmult0\widctlpar\adjustright {\b\fs28 \par }\pard \li3600\sl-360\slmult0\widctlpar\adjustright {\b\fs28 \hich\af4\dbch\af4\loch\f4 Travis Glenn}{\fs28 \par }\pard \li1080\widctlpar\tx4680\adjustright {\hich\af4\dbch\af4\loch\f4 Savannah River Ecology Lab\tab Department of Biological Sciences \par \hich\af4\dbch\af4\loch\f4 Drawer E\tab University of South Carolina \par \hich\af4\dbch\af4\loch\f4 Ai\hich\af4\dbch\af4\loch\f4 ken,SC 29802\tab Columbia, SC 29208 \par \hich\af4\dbch\af4\loch\f4 e-mail}{\hich\af4\dbch\af4\loch\f4 : }{\hich\af4\dbch\af4\loch\f4\tab e-mail: \par }\pard \widctlpar\adjustright { \par \par }\pard \fi720\widctlpar\adjustright {\hich\af4\dbch\af4\loch\f4 We use the following solutions & protocol to clean up our PCR products prior to cycle sequencing. In general, you will need about 10ng of purified PCR product per 100 bp of length (e.g., for a 500 bp PCR product, you need about 50 ng of template for seque \hich\af4\dbch\af4\loch\f4 n\hich\af4\dbch\af4\loch\f4 cing). The purpose of this protocol is to remove unused primers and dNTP's from the PCR. There are many alternative protocols (e.g., Qiagen columns, Microcon filters, etc.). This is the cheapest and most reliable in my hands. Note: If your PCR works reall \hich\af4\dbch\af4\loch\f4 y\hich\af4\dbch\af4\loch\f4 \hich\f4 well (i.e., 50+ ng of product per \u181\'b5\loch\f4 \hich\f4 L of PCR & no extra bands or primer dimers) then you can often simply use 0.5 to 1.0 \u181\'b5\loch\f4 L of your PCR reaction as the template (I also double the sequencing primer concentration, though I doubt it makes any difference).}{ \par }{ \par }\pard \widctlpar\adjustright {\b\ul \hich\af4\dbch\af4\loch\f4 St\hich\af4\dbch\af4\loch\f4 ock Solutions: \par }{\hich\af4\dbch\af4\loch\f4 1. 20% PEG, 2.5 M NaCl - Note: PEG takes 20+ min to go into solution when you make the initial solution. For 50mL, mix the following in a 50 mL conical: \par \hich\af4\dbch\af4\loch\f4 10.0 g PolyEthylene Glycol 8000 (MW = 6000 - 8000 is fine) \par \hich\af4\dbch\af4\loch\f4 7.3 g NaCl \par \hich\af4\dbch\af4\loch\f4 dH2O up to 45 mL -\hich\af4\dbch\af4\loch\f4 \hich\f4 shake & let PEG go into solution; I put it into the Boekel rocking incubator set to 37\u176\'a1\loch\f4 C \par \hich\af4\dbch\af4\loch\f4 after everything is in solution, fill with dH2O up to 50 mL. \par \hich\af4\dbch\af4\loch\f4 2. 80% EtOH in a 50mL conical or something similar. \par \hich\af4\dbch\af4\loch\f4 3. TLE = 10 mM Tris, 0.1 mM EDTA \par \par \hich\af4\dbch\af4\loch\f4 This protocol assum\hich\af4\dbch\af4\loch\f4 \hich\f4 es a 50 \u181\'b5\loch\f4 L PCR reaction and use of 0.2 mL thin walled tubes. If you have a different volume of PCR, then scale everything using the following proportions. If you use 0.5 mL tubes for PCR, then you don't need to use any new tubes. \par \hich\af4\dbch\af4\loch\f4 A) \hich\af4\dbch\af4\loch\f4 \hich\f4 Run out 5 \u181\'b5\loch\f4 L of PCR ont\hich\af4\dbch\af4\loch\f4 o an agarose gel to ensure the PCR reaction worked.}{ \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 \hich\f4 B) Add 50 \u181\'b5\loch\f4 L of PEG to a 0.5 mL tube. Transfer the remainder of the PCR to the tube with PEG and mix by pipetting up & down. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 \hich\f4 C) Let the the PCR + PEG incubate at 37\u176\'a1\loch\f4 C for 15 min. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 D) Place the 80% EtOH on \hich\af4\dbch\af4\loch\f4 ice. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 E) Centrifuge PCR + PEG at high speed (~15,000 x g) for 15 min. (at room temp.). \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 F) Using a P200 pipetter, pull off the supernatant & discard it. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 \hich\f4 G) Add 125 \u181\'b5\loch\f4 L of cold 80% EtOH to the tube. If you shoot the EtOH into the bottom of the tube, you must s\hich\af4\dbch\af4\loch\f4 pin for two minutes. If you place the EtOH onto the side of the tube, you can just let the tube sit for one minute. Using a P200 pipetter, pull off the supernatant & discard it. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 H) Repeat step G. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 I) Dry off the EtOH in a centrivap for 5-10 min, low heat, n\hich\af4\dbch\af4\loch\f4 o vacuum. There should be no trace (visible or by smell) of EtOH when done. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 \hich\f4 J) Dissolve the PCR product in 25 \u181\'b5\loch\f4 L of TLE. Pipette up & down several times to ensure the DNA has gone into solution. If you can let it sit for several minutes, that is also helpf \hich\af4\dbch\af4\loch\f4 ul. \par }{\fs16 \par }{\hich\af4\dbch\af4\loch\f4 \hich\f4 K) Run out 2-4 \u181\'b5\loch\f4 L onto an agarose gel for 10 min. to roughly quantify recovery. Use 20 and 100 ng of uncut lambda or plasmid DNA as a standard.}{ \par }}