Team:KAIT JAPAN/lab-note

LAB NOTE

AUGUST
August 15th
Presentation at Promega for sponsorship of reagents.
August 16th
Transformed designated parts into DH5α
BBa_I20270, R0040, J364000 ,J364001, J364002, J364003, J364004, J364005
August 17th
Broth culture shows no growth
August 18th
Transformed designated parts into DH5α
BBa_I20270, R0040, J364000, J364001, J364002, J364003, J364004, J364005
Orders from IDT
・IL-10 receptor
・STAT1
・JAK
・GFAP promotor
・HlyB
・HlyD
・IL-12
August 19th
Colonies were formed on all plates
But only a small number of colonies were found on the plates I20270, J364004 and J364005.
August 20th
Open campus
August 21st
Transformed I20270, J364004 and J364005 into DH5α.
August 22nd
Precultured all 8 of the parts, then cultured them for 16 to 18 hours.
August 23rd
Carried out measurements for interLab study
August 24th
Organized the data measured
interLab study completed!
August 25th
Received gBlocks gene fragments ordered from IDT
Add-on orders from IDT
Collaboration with Vincent Lim from National Yang-Ming University>
August 27th
PCR JAK5' and JAK3'
August 28th
PCR JAK5' and JAK3'
August 29th
PCR JAK5' and JAK3'
August 30th
1. Restriction digests and ligates JAK5' and JAK3'
2. PCR and gel purification of STAT1
3. PCR and gel purification of HlyD, HlyB and IL-12
August 31st
PCR of ligated JAK5'+3'(extension time 3:45)
SEPTEMBER
September 1st
PCR ligated JAK5' and 3' (extension time 3:45)
diluted 100x, 1000x, 10000x restriction digests and gel purificated STAT1.
restriction digests and gel purification of HlyD, HlyB
September 3rd
Preculture pSB1C3
HlyB＋D ligation→colony PCR
September 4th
Minipreparation→electrophoresis
HlyB＋D→electrophoresis
September 5th
Minipreparation→gel purification restriction enzyme treatment (EcoRI, SpeI)
restriction digests PCR-ed HlyB＋D (EcoRI, XbaI)→gel purification
September 6th
pSB1C3 and HlyD+HlyB ligation⇨transformation
PCR:
1. GFP+HlyA
2. HlyA+GFP
3. HlyA+IL-12
4. IL-12+HlyA
5. diluted TolC
September 7th
Gel purification of pSB1C3
PCR:
1. GFP+HlyA
2. HlyA+GFP
3. HlyA+IL-12
4. IL-12+HlyA
5. TolC
→Gel purification
September 8th
Restriction digests TolC and HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA
→Gel purification
→Ligation
→PCR for confirmation
September 11th
Ligates TolC and HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA
→Gel purification of PCR products
Preculturation of HlyD＋B
September 12th
1. Restriction digests TolC and HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA
→gel purification
2. Minipreparation of HlyD＋B
→Restriction digests
→Gel purification
→Ligation
→Transformation
September 13th
1. Restriction digests JAK
→Gel purification
2. Preculture of HlyD＋B＋TolC, HlyA+IL-12, HlyA+GFP, GFP+HlyA and IL-12+HlyA
September 14th
Miniprep HlyD＋B＋TolC, HlyA+IL-12, HlyA+GFP, GFP+HlyA and IL-12+HlyA
→We could not get the band wanted.
September 15th
Restriction digests HlyD＋B, JAK, pSB1C3 (EcoRI, SpeI)
September 17th
Third time of MetaGem
September 18th
PCR:
1. STAT
2. JAK
September 19th
Restriction digests STAT1 and JAK
→Gel purification
September 20th
1. Resuspend GFAPpromoter
→PCR
2. STAT1+JAK ligation
→PCR for confirmation
September 21st
1. PCR GFAPpromoter
2. STAT1+JAK gel purification
September 22nd
1. Restriction digests, ligation
i. STAT1 (Xba)
ii. IL-10receptor (SpeI)
→PCR for confirmation
2. GFAPpromoter gel purification
September 23rd
Ligation of STAT+IL-10receptor
→PCR for confirmation
September 24th
1. Preculturation of JAK inserted pSB1C3
2. PCR of TolC
September 25th
1. Minipreparation of JAK inserted pSB1C3
2. Restriction digests:ii. HlyA+IL-12, HlyA+GFP, GFP+HlyA and IL-12+HlyA (XbaI)
2. TolC (SpeI)
September 26th
1. Restriction digests of STAT+IL-10, JAK (EcoRI, SpeI)
→Gel purification
2. Gel purification of HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA and TolC
September 27th
Ligation, transformation
Ligation:
TolC+HlyA+IL-12, TolC+HlyA+GFP, TolC+GFP+HlyA and TolC+IL-12+HlyA
September 28th
Colony PCR
Completion of parts?
→Wanted band does not amplifies
Electrophoresis HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA
September 29th
HlyA+IL-12, HlyA+GFP, GFP+HlyA, IL-12+HlyA gel purification→✖️
OCTOBER
October 2nd
PCR⇨Gel purification:
1. GFP+HlyA
2. HlyA+GFP
3. HlyA+IL-12
4. IL-12+HlyA
October 3rd
PCR⇨Gel purification: TolC
1. TolC+GFP+HlyA
2. TolC+HlyA+GFP
3. TolC+HlyA+IL-12
4. TolC+IL-12+HlyA
October 4th
restriction digests→ligation→transformation of IL-10 and STAT1+JAK
October 5th
Pre-culture of IL-10+STAT1+JAK in PSB1C3
October 6th
1. Minipreparation of IL-10+STAT1+JAK in PSB1C3→PCR for confirmation
2. Restriction digests of
i. GFP+HlyA
ii. HlyA+GFP
iii. HlyA+IL-12
iv. IL-12+HlyA
then ligated with pSB1C3
October 7th
1. PCR: IL-10+STAT1+JAK→Wanted band does not amplifies
2. Transformation
i. GFP+HlyA
ii. HlyA+GFP
iii. HlyA+IL-12
iv. IL-12+HlyA
October 8th
Pre-culturation of:
1. GFP+HlyA
2. HlyA+GFP
3. HlyA+IL-12
4. IL-12+HlyA
October 9th
Ligates STAT1 and JAK with pSB1C3→Transformation
October 10th
Pre-culturation of STAT1+JAK in PSB1C3
October 11th
Mini-preparation of STAT1＋JAK in pSB1C3
October 12th
Restriction digests of STAT1＋JAK in pSB1C3
October 13th
Restriction digests of STAT1＋JAK in pSB1C3→Gel purification of minipreparated STAT1+JAK in pSB1C3
October 14th
1.Restriction digests of:
i. STAT1＋JAK (EcoRI, XbaI)
ii. IL-10receptor (EcoRI, SpeI)
2. Ligates restriction digested STAT1+JAK and IL-10receptor→Transformation
October 15th
Pre-culturation of:
1. GFAP+HlyD+HlyD+TolC+HlyA1
2. GFAP+HlyD+HlyD+TolC+HlyA3
3. pSB1C3+HlyA2
4. pSB1C3+HlyA1
5. IL-10+STAT+JAK
October 16th
Pre-culturation of:
1. GFAP+HlyD+HlyD+TolC+HlyA1
2. GFAP+HlyD+HlyD+TolC+HlyA3
3. pSB1C3+HlyA1
4. pSB1C3+HlyA3
5. IL-10+STAT+JAK
October 17th
Mini-preparation of:
1. GFAP+HlyD+HlyD+TolC+HlyA1
2. GFAP+HlyD+HlyD+TolC+HlyA3
3. pSB1C3+HlyA2
4. pSB1C3+HlyA4
5. IL-10+STAT+JAK
→Electrophoresis
PARTS COMPLETED!
October 18th
1. Restriction digests
i. HlyA1 (PstI, XbaI)
ii. HlyA3 (PstI, XbaI)
iii. pSB1C3 with HlyA2 (EcoRI, SpeI)
iv. pSB1C3 with HlyA4 (EcoRI, SpeI)
2. Ligation→Transformation
October 19th
Pre-culturation:
1. GFAP+HlyD+HlyD+TolC+HlyA1
2. GFAP+HlyD+HlyD+TolC+HlyA3
3. pSB1C3+HlyA2
4. pSB1C3+HlyA3
October 20th
1. Mini-preparation:
i. GFAP+HlyD+HlyD+TolC+HlyA1
ii. GFAP+HlyD+HlyD+TolC+HlyA3
iii. pSB1C3+HlyA2
iv. pSB1C3+HlyA4
→Restriction digest
October 21st
1. Restriction digests
i. GFAP+HlyD+HlyD+TolC (ＳpeI, PstI)
ii. HlyA2 (PstI, XbaI)
iii. HlyA4 (PstI, XbaI)
→Gel purification
→Ligation
→Transformation
October 22nd
Pre-culturation of:
1. GFAP+HlyD+HlyD+TolC+HlyA2
2. GFAP+HlyD+HlyD+TolC+HlyA4
October 23rd
Mini-preparation of:
1. GFAP+HlyD+HlyD+TolC+HlyA2
2. GFAP+HlyD+HlyD+TolC+HlyA4
→Restriction digests
PARTS COMPLETED!!
October 26th
Restriction digests:
1. IL-10+STAT+JAK
2. pCold
→Gel purification
October 27th
Ligation of IL-10+STAT+JAK with pCold
　　　　　
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