Team:HokkaidoU Japan/Notebook/H Stem/Construction

Notebook

Lab Documents

H-Stem Construction

Start
Mini-Prep
- Sun Aug 31
pSB1C3 vector
PCR
- Sun Aug 31, Mon Sep 1, Wed Sep 3, Thu Sep 4
R0010 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

R0040-B0034-E1010-B0015(plasmid) with NcoI_mRFP_F Tm:76.0℃ & XhoI_RBS_R Tm:68.2℃

B0015 (plasmid) with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
Digestion
- Sun Aug 31, Mon Sep 1, Wed Sep 3, Thu Sep 4
R0010 with SpeI

anti-sense with NcoI, XhoI

B0015 with XbaI
Primer Annealing
- Sun Aug 31, Thu Sep 4
stem_upstream_F Tm:71.4℃ & stem_upstream_R Tm:71.4℃

stem_downstream_F Tm:71.4℃ & stem_downstream_R Tm:71.4℃
Ligation
- Fri Sep 5
R0010 & stem-up & as-up

as-dn & stem-dn & B0015
PCR
- Fri Sep 5
R0010-stem-as with 100up_F Tm:62.6℃ & XhoI_RBS_R Tm:68.2℃

as-stem-B0015 with NcoI-mRFP_F Tm:76.0℃ & 200dn Tm:66.6℃
PCR
- Mon Sep 8, Tue Sep 9, Tue Sep 9
R0010-stem-as & as-stem-B0015 with 100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃
Digestion
- Wed Sep 10
Cut R0010-stem-as-stem-B0015 with EcoRI & PstI

Cut pSB1C3 with EcoRI & PstI
Ligation
- Wed Sep 10
Ligate R0010-stem-as-stem-B0015 with pSB1C3
Transformation
- Thu Sep 11
R0010-stem-as-stem-B0015(on pSB1C3)

5.0µL to DH5alpha;
Colony PCR
- Fri Sep 12, Sat Sep 13, Sat Sep 13
R0010-stem-as-stem-B0015(on pSB1C3) with

stem_up_F Tm:71.4℃ & stem_dn_R Tm:71.4℃

100up_F Tm:62.6℃ & 200dn_R Tm:66.6℃

EX_F Tm:70.4℃ & PS_R Tm:74.6℃
Complete!