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