"
Team:NCTU Formosa/Team
From 2012.igem.org
| Line 2,648: | Line 2,648: | ||
<li class="yellow e4"><p>Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li> | <li class="yellow e4"><p>Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1] </p></li> | ||
<li class="yellow e4"><p>PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK</p></li> | <li class="yellow e4"><p>PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK</p></li> | ||
| - | <li class="yellow e4"><p> | + | <li class="yellow e4"><p>SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K</p></li> |
</ul> | </ul> | ||
</div> | </div> | ||
| Line 2,836: | Line 2,836: | ||
<!---------------------------------------- week end ----------------------------------------> | <!---------------------------------------- week end ----------------------------------------> | ||
<!---------------------------------------- week start ----------------------------------------> | <!---------------------------------------- week start ----------------------------------------> | ||
| - | <div class="week | + | <div class="week"> |
<div class="day"> | <div class="day"> | ||
<div class="daybar"><p>22</p></div> | <div class="daybar"><p>22</p></div> | ||
Revision as of 15:01, 25 September 2012
Chang, Ting
Hello everyone! I am Zoe. I love to swim and drawing pictures.I am happy to join in this iGEM group. Many things I have learned from the process. It is truely a good experience to me forever. Zoe is the head of experiment group 1 which are responsible for assembling the first half circuits of our i-butanol-producing enzymes. She is the chief designer of our animation.
Hsieh, Hsin-Yun
Hello, She's Lisa Hsieh. She enjoys the process of the experiment with teammates. She likes to draw pictures. With a little perfectionism, it took her 2 days to finish team's mascot. She especially likes to drink a cup of hot cocoa after a long day work, and she believes cocoa can cure all diseases! Lisa worked with Zoe and Pearl to construct PSB II + ilvC and Ptet + B0032 + Gli1. She is the designer of our cute robot mascot.
Yu, Chun-Wei
Chun-Wei is a 3rd year biotechnology student at NCTU, love nature and playing guitar also love biology. He joined iGEM to get involved in the world-changing field of synthetic biology. Together with Jenny, Chun-Wei assembled the first two part of our enzyme series, and did the tolerance experiment. He is also an important role in our Human practice who trained students to be our human practice staffs.
Chen, Wei-Ting
Vicky Chen is a junior in National Chaio Tung University, major in bio-technology. She likes spending time in wet lab, discussing with her teammates. She enjoys eating foods ,shopping and traveling. Vicky assembled zif268 and alsS and optimized the growth condition in which E. coli can produce most i-butanol with Yu-Che.
Wu, Cheng-Hsuan
Wu, CHENG-HSUAN (English name: Max) is a third year Biological Science and Technology at NCTU. He enjoys playing basketball and going around the night markets. You can contact CHENG-HSUAN at max810327@hotmail.com.tw Max is the head of experiment group 2 which are responsible for assembling the second half circuits of our i-butanol-producing enzymes. Our instrument is mainly designed by him.
Lee, Chia-Jung
As a student majoring in biological science and technology in the third year in NCTU, Chia-Jung always want to know the logic and consequences behind things around him. He likes Chemistry and Biology particularly. In his free time, he likes playing tennis and training to improve his skill. Chia-Jung is the leader of 2012 NCTU-Formosa. He worked with group 2 to assemble biobrick and participated in summer camp as a TA.
Yu, Chieh-Cheng
Charlie white yu is a third-year student National Chiao Tung University who is majoring in Biological Science and Technology. He thought iGEM would be a great opportunity to further his knowledge of genetics, gain valuable research experience, and explore possible area of interest. Charlie is in charge of survey jobs and souvenir design. He was a menber of group 2 and also a TA in our summer camp.
Lin, I
Hi, my name is Lin I. I am from Taiwan and I major in Biotechnology in National Chiao Tung University. Playing basketball and listening classical music are my hobbies. It is really exciting and pleasant that I can be one of the 2012 iGEM team members. However, this is not all about me. If you have willing to contact me, maybe we will become friend! I is the “pro” of constructing biobricks in group 2. No matter how hard is it, I can built it. He also made our opening clip with Kyle.
Peng, Kai-Chun
Hi! I'm Kyle, a third year student contributing in Bio-technology at National Chiao Tung University of Taiwan. I'm interest in planting a variety of carnivorous plants which are so beautiful and enigmatic. I’m looking forward to meeting other school team. I hope it may be a real eye-opener for me. Besides being a member of group 2, Kyle played an important role in our summer camp. He edited handout written by TAs, designed courses, experiments and iGEM lectures. And he is the head of our video team which made our opening clip and animation.
Tsai, Lu-An
TSAI, Lu-An is a Qusai-Bachelor of NCTU, the adherent of Adeptus scientia and Preacher of capitalism. He loves the smell of napalm in the morning, the view of chaos in parliament. By using strange logic and weird metaphor, he can confuse the listeners, and Unfortunately himself. Besides being a member of group 2, Lu-An is the head of wiki group which made our beautiful team wiki. He also gave a lecture to students with pearl in summer camp.
Chung, Chia-Jung
Hello, I'm Pearl. I like to do experiments, and I go to lab rain or shine even on typhoon. Besides that, I like to chat with friend. I wear a big smile on my face .I like to practice cheerleading, too. Together with Lisa and Zoe, Pearl to construct PSB II + ilvC and Ptet + B0032 + Gli1. And she is the main designer of our T-shirt. She also gave a lecture to students with Lu-An in summer camp.
Cheng, Yu-Che
CHENG Yu-Che is a third year biotechnology student at National Chiao-Tung University. He's addicted to synthetic biology and molecular biology. So he may go this way in the future and keep playing table tennis which he is good at. Yu-Che assembled zif268 and alsS and optimized the growth condition in which E. coli can produce most i-butanol with vicky.
Hsu, Tzu-Yun
Jenny Hsu is a third-year student who is majoring in Biotechnology in National Chiao Tung University. She enjoys doing the experiments, cooking, and making handicraft. She thinks that attending 2012 iGEM is a good experience to her, and hopes she can make many friends in iGEM and share knowledge with each other. Jenny assembled the first two part of our enzyme series, and did the tolerance experiment with Chun-Wei. She also played an important role in summer camp. She connected our department for fund and gathered students to make our plan come true.
Calvin Hue
I’m a Photonics major at NCTU. I was overwhelmed with fun and experienced the power of Synaptic Biology during our cooperation. It’s also my pleasure to get involved in this project and make new friends. At the moment, I'm mainly working on the wiki site. I'm trying to make it eye-pleasing and user-friendly.
Tseng, Ching-Ping
He is a Professor of Biological Science and Technology at National Chiao Tung University, Hsinchu, Taiwan, R.O.C. Highest Education: Ph. D. Rutgers University, USA, 1991.Since 2001, Tseng has been a professor in NCTU, Taiwan. His current research interests include molecular biology, synthetic biology, applied microbiology, and bioengineering.
Lee, Hsiao-Ching
Received the B.S. degree and Ph.D. degree from the Department of Life Science at National Tsing Hua University, Hsinchu, Taiwan, R.O.C. in 1999 and 2005 respectively. From 2005 to 2007, she worked as a postdoctoral research position at NTHU. She is now an Assistant Professor with the Department of Biological Science and Technology at National Chiao Tung Universit. Her current research interests include molecular biology, synthetic biology and bioinformatics.
Huang, Hsien-Da
Dr. Huang is a Professor and the current Chairman of Department of Biological Science & Technology, Institute of Bioinformatics and Systems Biology of National Chiao Tung University, Hsinchu, Taiwan R.O.C. His current research include microRNA regulation and protein post-translational modification databases building and dry-lab tools development.
Yu-Pei Chen
Received the Ph.D. degree from the Department of Biological Science and Technology at National Chiao Tung University, Hsinchu, Taiwan, R.O.C. in 2008. From 2008 to 2012, he worked as a postdoctoral research position in Department of Biological Science and Technology and Mechanical Engineering at NCTU. His current research interests include molecular biology, biochemistry and fungi secondary metabolite.
Tseng, De-Jung
I’m De-Jung Tseng, a fourth grade student of National Chiao Tung University. Talented at fine art and literary creation , I have high curiosity of everything in daily life and plenty of ideas. Believing in the innovation of science ,I'm passionate to have fun in iGEM 2012. The limit is the mark which would be overcome next. I designed our beautiful souvenir.Wish you all would like it.
Malvin Jefri
An International student in NCTU, majoring in Biological Science and Technology. I'm a badminton freak who loves watching Korean serial movies and Korean boy bands. It's my pleasure to join IGEM competition to taste what's so called teamwork in doing experiment as a team. There's so much sweet n sour moment during these period. Last, I'm just An ordinary Indonesian boy who does ordinary stuffs but always planned to go beyond my limit.
Host Lab
In this year preparation, most of our experiments were done in the Applied Microbiology & Biochemistry Lab, which also known as CPT Lab named after supervisor prof. Ching-Ping Tseng. CPT Lab locates at National Chiao Tung University (Boai campus), Hsinchu, Taiwan, R.O.C.
Come visit the website of our lab: http://life.nctu.edu.tw/~cptlab/Home.html
Notes
March 2012
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
mini-prep of cultivated AlsS, pSB1K3, pSB1A3, and pSB1C3 E.coli (G2)
digestion : [Zif268] ES & [AlsS] XP & [pSB1C3] EP (G2)
ligation : insert[Zif268] ES & [AlsS] XP/Vector[pSB1C3] EP (G2)
Transformation of B0034
Cultivation of transformed E.coli on LBA plate
Transformation of Zif268+AlsS+pSB1C3 in DH5-alpha (G2)
Point mutation HivC (PCR, DPN1 digest, TA cloning, transform) (G2)
PCR of point mutation HivC (G2)
PCR of insert fragment [Zif268+AlsS] (G2)
electrophoresis of insert fragment [Zif268+AlsS]----NOT OK (G2)
Single colony isolation from pLac LBA plate, and cultivation of pLac E.coli in liquid LBA
Single colony isolation from pSB1K3 LBK plate, and cultivation of pSB1K3 E.coli in liquid LBK
mini-prep of cultivated pLac & pSB1K3 E.coli
digestion : [pSB1K3] EP
Single colony isolation from Zif268 LBA plate, and cultivation of Zif268 E.coli in liquid LBA
Single colony isolation from AlsS LBA plate, and cultivation of AlsS E.coli in liquid LBA
mini-prep of cultivated Zif268 Ecoli & AlsS Ecoli
digestion :[Zif268] ES/[AlsS] XP
April 2012
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]
transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate
digestion : [pSB1K3] EP
digestion : [pSB1K3] EP
PCR of insert fragment [Zif268+AlsS] OK
DNA Sequencing OK
ligation : point mutation HivC
TA cloning : point mutation HivC
Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K
mini-prep of cultivated Zif268+ AlsS E. coli
transformation of point mutation HivC and cultivation on LB-A plate
transformation of B0034 and cultivation on LB-A plate
Single colony isolation from HivC LB-A plate, and in liquid LB-A
Single colony isolation from B0034 LB-A plate, and in liquid LB-A
mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli
digestion : Zif268+AlsS [XP]
Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated ilvD E. coli
digestion : ilvD [EP] & pSB1K3 [EP]
transformation of 37℃ RBS and cultivation on LB-C plate
Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A
digestion : B0034 [SP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]
mini-prep of cultivated Hivc E. coli
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
transformation of HivC and cultivation on LB-A plate
PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing NOT OK
digestion : B0034 [SP] & Zif268+AlsS [XP]
gel extraction
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A
SCI from ilvD LB-K plate, and cultivation in liquid LB-K
PCR of insert fragment [B0034+Zif268+AlsS] NOT OK
transformation of B0034+Zif268+AlsS and cultivation on LB-A plate
mini-prep of cultivated B0034 and ilvD E. coli
Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated HivC E. coli
digestion : B0034 [SP] & ilvD [XP]
digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]
ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]
ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]
transformation of HivC+ilvD and cultivation on LB-A & LB-C plate
PCR of insert fragment [B0034+Zif268+AlsS] NOT OK
transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate
PCR of insert fragment [HivC+ilvD] OK
DNA sequencing NOT OK
Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C
mini-prep of cultivated & pSB1C3 E. coli
May 2012
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
digestion : pLac [EP] & pSB1K3 [EP]
ligation :insert pLac [EP]/Vector pSB1K3 [EP]
transformation of pLac+pSB1K3 and cultivation on LB-K plate
PCR of insert fragment [pLac+pSB1K3] OK
DNA Sequencing OK
transformation of B0034_new and cultivation on LB-A plate
Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034 E. coli
digestion : B0034 [SP]
ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]
transformation of B0034+Zif268+AlsS
cultivation on LB-A plate
PCR of insert fragment [B0034+Zif268+AlsS]-----self ligation
transformation of 37℃ RBS and cultivation on LB-C plate
transformation of 37℃ RBS and cultivation on LB-Kplate
Single colony isolation from 37℃ RBS LB-K plate, and cultivation in liquid LB-K
digestion : B0034 [ES] & pSB1C3 [EP]
mini-prep of cultivated 37℃ RBS E. coli
ligation :insert B0034 [ES] & pSB1C3 [EP]/Vector pSB1C3 [EP]
transformation of B0034+Zif268+AlsS and cultivation on LB-C plate
digestion : pLac [ES] & pLac [SP]
digestion : B0034 [SP] Kr (gel extraction) , B0034 [SP] Ar (gel extraction) , 37℃ RBS [XP] & HivC [XP]
ligation : (1. 2 parts) insert HivC [XP], Vector B0034+pSB1K3 [SP]
ligation : (2. 3 parts) insert HivC [XP], Vector B0034+pSB1A3 [SP]
PCR of insert fragment [B0034+Zif268+AlsS] OK
DNA Sequencing OK
transformation of B0034+ HivC and cultivation on LB-A & LB-K plate
Single colony isolation from pSB1A3 LB-A plate, and cultivation in liquid LB-A
Single colony isolation from B0034+Zif268+AlsS LB-C plate, and cultivation in liquid LB-C
Single colony isolation from B0034+HivC LB-A & K plate, and cultivation in liquid LB-A & K
mini-prep of cultivated pSB1A3 E. coli & B0034+Zif268+AlsS
digestion: pSB1A3 [EP] & B0034+Zif268+AlsS [XP]
ligation: (1.3 parts)insert pLac [ES] & B0034+Zif268+AlsS [XP], Vector pSB1A3 [EP]
ligation: (2.2 parts)insert B0034+Zif268+AlsS [XP], Vector pLac+pSB1K3 [SP]
transformation of pLac+B0034+Zif268+AlsS and cultivation on LB-A plate & LB-K plate
mini-prep of cultivated B0034+HivC (Ar & Kr)
PCR of insert fragment [pLac+B0034+Zif268+AlsS] OK
DNA Sequencing 3 parts OK
digestion : 37℃ RBS [XP] , ilvD [ES] & pSB1C3 [EP]
digestion : HivC+ilvD-3,18 [EP]
ligation :insert ilvD[ES] & 37℃ RBS [XP], Vector pSB1C3 [EP]
Single colony isolation from pLac+B0034+Zif268+AlsS LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated pLac+B0034+Zif268+AlsS E. coli
transformation of ilvD+37℃ RBS and cultivation on LB-C plate
PCR of insert fragment [ilvD+37℃]-----OK
DNA Sequencing OK
Single colony isolation from PBSII+ilvC LB-K plate, and cultivation in liquid LB-K
ligation : insert HivC [XP], vector B0034+pSB1A3 [SP]
mini-prep of cultivated PBSII+ilvC E. coli
digestion : pSB1C3 [EP] & PBSII+ilvC [XP]
ligation : insert B0034 [ES] & PBSII+ilvC [XP], vector pSB1C3 [EP]
transformation of B0034+PBSII+ilvC and cultivation on LB-C plate
transformation of B0034+HivC and cultivation on LB-A plate
June 2012
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
PCR of insert fragment [B0034+PBSII+ilvC] OK!
DNA Sequencing OK!
mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli
mini-prep of cultivated HivC+ilvD-12,20 E. coli
digestion : HivC+ilvD-12,20 [XP]
Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)
digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]
ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]
ligation : insert HivC [EP]/vector pSB1C3 [EP]
transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate
PCR of insert fragment [B0034+ HivC] & [HivC] OK
Design the primer for B0034+PBSII+ilvC point mutation (pm)
Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C
mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]
transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed
DNA Sequencing B0034+HivC & HivC OK
PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK
Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A
Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli
Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better
PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK
DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK
PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK
digestion: pLac+B0034+Zif268+AlsS [DPn1]
transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate
Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli
digestion : pLac+B0034+Zif268+AlsS (pm) [EP]
DNA sequencing OK
PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK
digestion : B0034+ PBSII+ilvC [DPn1]
transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail
PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK
digestion : B0034+ PBSII+ilvC [DPn1]
transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate
PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK
Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C
July 2012
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli
digestion : B0034+ PBSII+ilvC (pm) [EP]
DNA sequencing OK
digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR
digestion : pSB1K3 [EP]
ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]
transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate
Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR
Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]
PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK
SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K
mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli
DNA sequencing OK
culture condition test: activation DH5αovernight
transfer to new medium(1/100), OD0.2 start counting culture time
transfer to 30゜C and 27゜C
transfer to 30゜C and 27゜C
transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate
PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK
Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]
Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]
transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)
PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK
Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A
mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli
Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]
ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]
transformation of G1+G2,and cultivation on LB- C plate failed
ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]
transformation of G1+G2,and cultivation on LB- C plate
Digestion: G2’(B0034+HIVC+ilvD) [XP]
ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]
PCR of insert fragment [kivD+B0015]
transformation of G1+G2’, and cultivation on LB- C plate failed
Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C
sample and do GC
mini-prep of cultivated kivD+B0015 E. coli
Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]
Digestion: G2’ [DPn1]
transformation of G2’, and cultivation on LB- A plate
Digestion: kivD+B0015 [EP] (checking bp----failed)
Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A
