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From 2012.igem.org

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 P2
ADN: TirI-PCR P2-P36 (08.08) + TirI PCR P1-P4 (10.08)

pCS2+

Primers: P7+P8
ADN:A1
=> These PCR didn't work

New PCR

-TirI + P1-P4 with or without DMSO
-TirI + P2-P36 with or without DMSO
-pCS2+ mCherry + P7-P8
-pCS2+ TC + P7-P8

Cloning pSB1C3/IRES, pSB1C3/IaaM, pSB1C3/IaaH, pCS2+/IaaH (to biobrick pCS2+)

Restriction with EcoRI and PstI for pSB1C3 (K515100) then gel extraction of the backbone
Restriction 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

Transformation into Top10 E. coli

10ul sample + 50ul competent cells 250ul SOC medium pSB1C3/IRES
pSB1C3/IaaM
pSB1C3/IaaH
pSB1C3/IaaH-IRES
pCS2+/IaaH
pCS2+/IRES

Plates

pSB1C3 chloramphenicol resistant
pCS2+ ampicillin resistant

Tuesday, 14th August

Colony PCR

Positive Top 10 colonies:
pSB1C3/IRES
pSB1C3/IaaH-IRES
-> the result of the colony PCR is not as expected, IRES and IaaH are not present in pSB1C3

Thursday, 16th August

PCR

pCS2+ samples J5, J6, J18, J21, purif 13/08 with Primers: P7&P8
IaaH with primers 34 and 20, IaaM with primers 35 and 22, IRES with primers 31 and 32