Team:EPF-Lausanne/Notebook/Cloning Strategy

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=Cloning strategy=
=Cloning strategy=
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===13.07.09===
 
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Restriction enzymes  on [http://www.neb.com/nebecomm/products/category1.asp?#2 Biolabs website]
 
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and [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp clevage oligonucleotides]
 
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'''TRP promoter biobrick strategy'''
 
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:* Problem to overcome:
 
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: SpeI sites on Trp promoter sequence and it's an upstream part which has to be cut with ES.
 
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:* Strategy:
 
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: PCR: Forward primer having E and X sites and Reverse primer NheI.
 
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: Digest Trp promoter with E and NheI.
 
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: Digest plasmid with E and X.
 
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: Ligation -> E site is recreated; X and NheI have compatible ends so ligation is possible and the site is destroyed (mixted site).
 
===14.07.09===
===14.07.09===

Revision as of 07:47, 28 July 2009



Contents


Cloning strategy

14.07.09

Primers designed for LOVTAP read-out and RBphP project.

15.07.09

16.07.09

17.07.09

20.07.09

21.07.09

One useful website to know the restriction sites of enzymes:
http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez

The restriction site used were:
EcoRI GAATTC
XbaI TCTAGA
SpeI ACTAGT
PsiI TTATAA

Design the primers for the 2 step-PCR: the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream. In the second step we only introduce the E-X restriction sites upstream and the SP downstream.

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23.07.09

24.07.09

27.07.09

28.07.09

29.07.09

30.07.09

31.07.09

August

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