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Team:Evry/Notebook/w10
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Directly after fertilization of xenopus tropicalis eggs: injection of pNHK60 (plasmid with Tir1 and GFP-aid)<br> | Directly after fertilization of xenopus tropicalis eggs: injection of pNHK60 (plasmid with Tir1 and GFP-aid)<br> | ||
The production of GFP is expected into eggs<br> | The production of GFP is expected into eggs<br> | ||
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| + | <h3>Transformation in <i>Top10 E. coli</i> </h3> | ||
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Revision as of 14:34, 13 August 2012
Weeks:
| June | July | August | September | October | November | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Week 10: 13th August - 19th August
Monday, 13th August
Tadpoles injection (pNHK60)
Preparation of pNHK60 dilution(phenol/chloro extraction of Friday, 10th August)Tube A12 (50ng/ul): 1,695ul A11 + 73,305ul H2Odd
Tube A13 (25ng/ul): 25ul A12 + 25ul H2Odd
PCR
TirI
Primers: P1 and P2ADN: TirI-PCR P2-P36 (08.08) + TirI PCR P1-P4 (10.08)
pCS2+
Primers: P7+P8ADN:A1
Cloning pSB1C3/IRES, pSB1C3/IaaM, pSB1C3/IaaH, pSC2+/IaaH (to biobrick pCS2+)
Restriction with EcoRI and PstI for pSB1C3 (K515100) then gel extraction of the backboneRestriction with EcoRI and PstI for product of PCR: IRES, IaaH, IaaM and pCS2+ then purification with PCR clean up kit
Then ligation of pCS2+ with IaaH, pSB1C3 with IRES, pSB1C3 with IaaM and pSB1C3 with IaaH
Injection of PNHK60 into Xenopus eggs
Directly after fertilization of xenopus tropicalis eggs: injection of pNHK60 (plasmid with Tir1 and GFP-aid)The production of GFP is expected into eggs
