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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

IaaH with primers 34 and 20, IaaM with primers 35 and 22, IRES with primers 31 and 32
Tir I "mega" PCR: with Tir I P1-P4 and TirI P2-P36 PCR products + P1 and P2 primers

pSC2+

PCR pSC2+ samples J5, J6, J18, J21, purif 13/08 with Primers: P7&P8

(1) PCR sample J5
(2) PCR sample J6
(3) PCR sample J18
(4) PCR sample J21
(5) PCR purif 13/08








Gel extract 1, 2, 3 and 5. Elution in 20ul in EB
(1) 10,2 ng/ul
(2) 45,6 ng/ul
(3) 20,3 ng/ul
(4) .
(5) 25,6 ng/ul

Ligation
RFP-Dig16/08-WR: 5ng/ul
IaaH-Purif13/08-KC: 84,5ng/ul
pSC2+-n°5-gelextract-KC: 25,6ng/ul

Ligation pSC2+/RFP
H2Odd 1,92ul
Buffer 1,5ul
pSC2+ 3,91ul
RFP 6,67ul
T4 ligase 1ul

Ligation pSC2+/IaaH
H2Odd 7,49ul
Buffer 1,5ul
pSC2+ 3,91ul
IaaH 1,1ul ==> no more material so 0,5ul
T4 ligase 1ul

Overnight 16°C

Biobricking Promoters : Flk-1 & CarA

Friday, 17th August

Transformation

Ligations 16/08
10ul ligation + 50ul top10 competent cells
500ul LB pre-warmed
No centrifugation before spread 150ul on plate AMP
Incubation until the end of day at 37°C then RT for the week end

Gels

Gel extraction:

Size is ok for IaaH, IaaM and IRES.