Protocol/17

From 2010.igem.org

(Difference between revisions)
(New page: {{Team:Alberta/Head}} {{Team:Alberta/navbar|project=selected}} {{Team:Alberta/beginLeftSideBar}} {{Team:Alberta/endLeftSideBar}} {{Team:Alberta/beginRightSideBar}} {{Team:Alberta/endRig...)
Line 13: Line 13:
Protocol 17: PCR
Protocol 17: PCR
-
'''Before you start:'''
 
-
* KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
 
-
* Reserve a thermocycler and check what size of tubes it takes.
 
-
* Making a master mix conserves expensive reagents, so try to always use one.
 
-
* You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
 
-
* DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
 
-
'''Protocol:'''
+
Procedure:
-
*PCR buffer         5ul
+
* Before you start:
 +
** KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
 +
** Reserve a thermocycler and check what size of tubes it takes.
 +
** Making a master mix conserves expensive reagents, so try to always use one.
 +
** You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
 +
** DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
 +
*To add into a tube:
 +
{|
 +
|PCR buffer || 5ul
 +
|-
 +
|10uM dNTPs || 1ul
 +
|-
 +
|50uM MgCl2 || 2ul
 +
|-
 +
|Forward primer || 2.5ul
 +
|-
 +
|Reverse primer || 2.5ul
 +
|-
 +
|1ng Template || 1ul
 +
|-
 +
|Taq polymerase || 0.5ul
 +
|-
 +
|MilliQ water || 35.5ul
 +
|-
 +
|TOTAL || 50ul
-
*10uM dNTP's        1ul
 
-
 
-
*50uM MgCl2          2ul
 
-
 
-
*forward primer      2.5ul
 
-
 
-
*reverse primer      2.5ul
 
-
 
-
*1ng template DNA    1ul
 
-
 
-
*Taq polymerase      0.5ul
 
-
 
-
*MilliQ (H2O)        35.5ul (to make total volume 50ul)
 
-
 
-
TOTAL              50ul
 
[[Team:Alberta/Notebook/protocols| Back]]
[[Team:Alberta/Notebook/protocols| Back]]
{{Team:Alberta/endMainContent}}
{{Team:Alberta/endMainContent}}

Revision as of 02:17, 26 October 2010

TEAM ALBERTA

Protocol 17: PCR


Procedure:

  • Before you start:
    • KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
    • Reserve a thermocycler and check what size of tubes it takes.
    • Making a master mix conserves expensive reagents, so try to always use one.
    • You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
    • DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
  • To add into a tube:
PCR buffer 5ul
10uM dNTPs 1ul
50uM MgCl2 2ul
Forward primer 2.5ul
Reverse primer 2.5ul
1ng Template 1ul
Taq polymerase 0.5ul
MilliQ water 35.5ul
TOTAL 50ul


Back

Retrieved from "http://2010.igem.org/Protocol/17"