|
|
| Line 1: |
Line 1: |
| ==Overview==
| |
|
| |
|
| Replace this sentence with a brief description of the protocol and its goal.
| |
|
| |
| ==Materials==
| |
|
| |
| For a 50 μL PCR reaction:
| |
|
| |
| * 35 μL H<sub>2</sub>O
| |
| * 5 μL 10X PCR buffer
| |
| * 5 μL 2mM dNTPs (each)
| |
| * 1.5 μL 50mM MgCl<sub>2</sub>
| |
| * 1 μL 50μM sense primer
| |
| * 1 μL 50μM antisense primer
| |
| * 1 μL 5nM DNA template
| |
| * 0.5 μL TAQ DNA polyermerase
| |
|
| |
| ==Procedure==
| |
| # In a PCR tube, mix the components on ice in the order they are listed above.
| |
| # Perform thermocycling program
| |
| ## 95 °C 5 min
| |
| ## 95 °C 30 s
| |
| ## T<sub>H</sub> 30 s
| |
| ## 72 °C 1 min for each 1 kb PCR product
| |
| ## Repeat steps 2-4 a total of 12-36 times (24 is standard).
| |
| ## 72 °C 5 min
| |
| ## 12 °C hold
| |
|
| |
| ==Notes==
| |
| Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
| |
| #List troubleshooting tips here.
| |
| #You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
| |
| #Anecdotal observations that might be of use to others can also be posted here.
| |
|
| |
| Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
| |
|
| |
| ==References==
| |
| '''Relevant papers and books'''
| |
| <!-- If this protocol has papers or books associated with it, list those references here.-->
| |
| <!-- See the [[OpenWetWare:Biblio]] page for more information, but this format doesn't work currently.
| |
| <biblio>
| |
| #Goldbeter-PNAS-1981 pmid=6947258
| |
| #Jacob-JMB-1961 pmid=13718526
| |
| #Ptashne-Genetic-Switch isbn=0879697164
| |
| </biblio>-->
| |
| <!-- Try the [[Template:FormatRef|FormatRef template]]-->
| |
| #{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
| |
| #{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
| |
| #{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164
| |
|
| |
| ==Contact==
| |
| *Who has experience with this protocol?
| |
|
| |
| or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].
| |
|
| |
| <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
| |
|
| |
| <!-- Move the relevant categories above this line to tag your protocol with the label
| |
| [[Category:Protocol]]
| |
|
| |
| [[Category:Needs attention]]
| |
|
| |
| [[Category:In vitro]]
| |
|
| |
| [[Category:In vivo]]
| |
|
| |
| [[Category:In silico]]
| |
|
| |
| [[Category:DNA]]
| |
|
| |
| [[Category:RNA]]
| |
|
| |
| [[Category:Protein]]
| |
|
| |
| [[Category:Chemical]]
| |
|
| |
| [[Category:Escherichia coli]]
| |
|
| |
| [[Category:Yeast]]
| |
