"
Team:HokkaidoU Japan/Notebook/Length/Future Work
From 2014.igem.org
(Difference between revisions)
| Line 140: | Line 140: | ||
</ul> | </ul> | ||
<div class="step-recipe"> | <div class="step-recipe"> | ||
| - | R0040-B0034-E1010-B0015 with Ptet_XhoI_F Tm:75.1℃ & mRFP_400dn_R Tm:67.0℃ KOD plus Neo | + | R0040-B0034-E1010-B0015 with Ptet_XhoI_F Tm:75.1℃ <br>& mRFP_400dn_R Tm:67.0℃ KOD plus Neo |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 00:43, 18 October 2014
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.
