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Team:HokkaidoU Japan/Notebook/Length/Future Work
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Latest revision as of 15:33, 9 September 2015
Notebook
Lab Documents
Future Work
-
Start
-
prePCR
R0040-B0034-E1010-B0015 with Ptet_XhoI_F Tm:75.1℃ & mRFP_400dn_R Tm:67.0℃ KOD plus Neo -
PCR adding Klenow
R0040-B0034-E1010-B0015t 20cycles, 35cycles annealing 40℃ -
Gel extraction
inserts -
Ethanol precipitation
inserts -
Digestion
Cut inserts and pHN1257 with NcoI, XhoI(using 10×CutSmart) -
Gel extraction
inserts, pHN1257 -
Ethanol precipitation
inserts, pHN1257 -
Dephosphorylation
Antactic phosphorylate inserts (using 10×Antarctic phosphatase buffer) 37℃ 35min 65℃ 10min -
Ligation
Ligate inserts with pHN1257 -
Transformation
ligation products 5.0µL DNA to DH5α -
Colony PCR
sequenses are unknown because of randomizing(on pHN1257) pHN1257_up_F Tm:60.1℃ & as_RBS_XhoI_R Tm:68.2℃ Kapa-Taq -
Complete!
We could not get hoped result though we got some colonies. However, stem region has a problem that PCR is not performed well. It is possible that the constructs were complete.
