"
Team:KAIST Korea/Notebook Labnote/2012 9
From 2012.igem.org
2012 KAIST Korea
Mail : kaist.igem.2012@gmail.com
Twitter : twitter.com/KAIST_iGEM_2012
Facebook : www.facebook.com/KAISTiGEM2012
Notebook : Labnote-September
Labnote
SeptemberSeptember 1st 2012
PACKMAN
fdh pBAD, fdh pTrc colony PCR check
Results
Discussion
To check the presence of FDH gene, we picked the colony from the plate and did colony PCR. For all the colonies we picked, we confirmed that correct size of FDH is present.
Flip FlopBxb1_GTG_pBAD cloning with remaining DNAs
Remaining Bxb1_GTG insert was cloned into pBADmycHisC vector. And electrotransformed into MG1655.
Back to the Calendar
Back to the CalendarSeptember 2nd 2012
Flip FlopBxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc miniprep for double transformation, Bxb1_GTG_pBAD colony inoculation and vector miniprep NcoI single cut check.
Results
Discussions
All the vectors showed right sizes. But Bxb1_GTG_pBAD colony #1 through 3 have undesired bands. They may be the supercoiled vector.
Bxb1_ATG_pTrc, pBAD, Bxb1_GTG_pTrc double transformation into pPoC and pPoCpi
Back to the Calendar
September 3rd 2012
Flip FlopBxb1 double transformants vector miniprep & PCR check
Results
Discussions
Seemed to every colonies have appropriate vectors.
Back to the Calendar
September 4th 2012
No Special Event!
Back to the Calendar
September 5th 2012
No Special Event!
Back to the Calendar
September 6th 2012
No Special Event!
Back to the Calendar
September 7th 2012
No Special Event!
Back to the Calendar
September 8th 2012
No Special Event!
Back to the Calendar
September 9th 2012
No Special Event!
Back to the Calendar
September 10th 2012
Flip FlopBxb1_GTG_pBAD single cut check with different enzyme
Single cut with SnaBI
Results
No desired size(5.6kb) vector appeared.
Back to the Calendar
Flip Flop
Flip Flop
Flip Flop
Flip Flop
September 11th 2012
No Special Event!
Back to the Calendar
September 12th 2012
Flip FlopBxb1_GTG_pBAD colony #4~7 inoculation, miniprep and single cut check
Single cut with SnaBI
Single cut with SnaBI
Results
Cloning completed
Back to the Calendar
September 13th 2012
No Special Event!
Back to the Calendar
September 14th 2012
No Special Event!
Back to the Calendar
September 15th 2012
Flip FlopBBa_C0060, BBa_C0061, BBa_C0062 transformation into TOP10
Templates for Auto-regulated FlipFlop project.
Back to the Calendar
September 16th 2012
Flip FlopBBa_C0060, BBa_C0061, BBa_C0062, bFMO inoculation and enzyme cut check
BBa_C0060, BBa_C0061, BBa_C0062: from colony, EcoRI single cut
Back to the Calendar
- BBa_C0060: 2859bp
- BBa_C0061: 2688bp
- BBa_C0062: 2826bp
- bFMO: from cell stock, EcoRI single cut
September 17th 2012
Flip FlopTemplate preparation for OE PCR
Promoter construct: 210bp
Results
Promoter PCR failed
Back to the Calendar
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
Flip Flop
September 18th 2012
Flip FlopTemplate preparation for OE PCR
Results
Terminator PCR failed
Back to the Calendar
September 19th 2012
Flip FlopTemplate preparation for OE PCR
Terminator: 179bp
Results
→ Gel extracted
Back to the Calendar
September 20th 2012
Flip FlopTemplate preparation for OE PCR
Bxb1 Xis: 777bp
Results
bFMO PCR w/ various condtions
Bxb1_Xis-pGEM_B1 transfomation
Synthesized gene – Bxb1_Xis arrived and transformed into TOP10
Back to the Calendar
September 21st 2012
No Special Event!
Back to the Calendar
September 22nd 2012
Flip FlopOverlapping extension PCR
Results
→ Gel extracted
Back to the Calendar
September 23rd 2012
Flip FlopOverlapping extension PCR-Nested PCR
Results
Discussions
There was too little full construct nested. We think this is due to primer interfering by its LVA homology.
Overlapping extension PCR 2
Results
Discussions
Bands were more intense than those of first trial.
Back to the Calendar
September 24th 2012
Flip FlopOverlapping extension PCR Gel Extraction
Results
Back to the Calendar
P1, PI construct
Results
Full fragment for pAuto is overlapped. The right size(3794bp) fragments are gel extracted.
September 25th 2012
No Special Event!
Back to the Calendar
September 26th 2012
Flip FlopFull pAuto and pAutoSimple Overlapping PCR
Results
Discussion
The band intensity is too weak.
Back to the Calendar
pAutoIntegrase Overlapping PCR
Results
Full construct(2.5kb) for pAutoIntegrase is overlapped. The products are pcr purified and performed nested-pcr. 4th lane is the OE pcr product with rearranged template concentration.
Template preparation of terminator
Results
Terminator template for OE pcr is re-amplified with pfu-X DNA polymerase. The products are pcr purified.
September 27th 2012
No Special Event!
Back to the Calendar
September 28th 2012
No Special Event!
Back to the Calendar
September 29th 2012
Flip FlopTemplate preparation of bFMO (for pAuto and pAutoSimple)
Results
To find out the right temperature for primer binding, we performed gradient pcr from 45℃~55℃.
But, all the pcr products showed non-specific binding of primers, and we selected the most proper temperature.
Back to the Calendar
Overlapping extension PCR Gel Extraction
Results
September 30th 2012
Flip FlopTemplate vector preparation – double digestion check with EcoRI and PstI
Results
Vector(BBa_J04450-pSB4A5) which will be used for cloning of pAutoIntegrase.
Back to the Calendar
bFMO template amplification with optimized condition
Results
pAutoIntegrase cloning check
Results
PCR amplified from prepped vector
