Difference between revisions of "Lowering linear plasmid concentrations"

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This is a protocol made by the Lui lab to lower the the copy number of pGKL1 using a strain (replace strain here) our lab created
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
# 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
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.
'''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.
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
<!-- 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
*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:Needs attention]]
[[Category:In vitro]]
[[Category:In vivo]]
[[Category:In silico]]
[[Category:Escherichia coli]]

Latest revision as of 13:53, 4 September 2013