PCR Amplification of DNA: Difference between revisions

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==SOP==

*[[SOP - Electrophoresis]]

==Materials==

*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)

*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 0.4uM-1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)

*dNTPs ~~– dilute to 2 mM each in water~~, ~~make single use aliquots (50 uL aliquots~~..~~. 10uL of each dNTP, 460uL water)~~

*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye https://www.thermofisher.com/order/catalog/product/K1081?ICID=search-product#/K1081?ICID=search-product

*~~Template – generally 1uL or less of a plasmid miniprep~~

*RNAse-Free water - comes in DreamTaq kit

*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

==Protocol==

*Use the following volumes per reaction

::*~~Buffer,~~ 5 uL ~~of 10X buffer (Dave's fridge)~~

::*12.5 uL DreamTaq

::*~~Primers, 10uL~~ of 1uM stock solution in water (both primers combined)

::*7.5 uL RNAse-Free water

~~::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)~~

::*5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))

~~::*Sterile water, 28 uL~~

::*~~Template 1 uL~~

==PCR Program==

:~~:*Polymerase 1 uL (turbo pfu found in "enzymes" box~~ in ~~freezer)~~

*For animals: use programs denoted in [[Genotyping Program]]

*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:

@@ Line 1: / Line 1: @@
+==SOP==
+*[[SOP - Electrophoresis]]
 ==Materials==
-*Primers – order from IDT prediluted to 100 mM in TE.  Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
+*Primers – order from IDT prediluted to 100 mM in TE.  Make working solution of 0.4uM-1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
-*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
+*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye  https://www.thermofisher.com/order/catalog/product/K1081?ICID=search-product#/K1081?ICID=search-product
-*Template – generally 1uL or less of a plasmid miniprep
+*RNAse-Free water - comes in DreamTaq kit
-*Polymerase – use Pfu Turbo for cloning and Taq for noncloning.  Use appropriate buffer.
 ==Protocol==
 *Use the following volumes per reaction
-::*Buffer, 5 uL of 10X buffer (Dave's fridge)
+::*12.5 uL DreamTaq
-::*Primers, 10uL of 1uM stock solution in water (both primers combined)
+::*7.5 uL RNAse-Free water
-::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
+::*5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))
-::*Sterile water, 28 uL
-::*Template 1 uL
+==PCR Program==
-::*Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)
+*For animals: use programs denoted in [[Genotyping Program]]
 *Run PCR Program (approx 3.5 to 4 hours).  Normally use touchdown PCR ('''DAVETD''') as follows: