"
Team:KAIST Korea/Notebook Labnote/2012 6
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-June
Labnote
JuneJune 1st 2012
Vector mini-prep: Moth_gene carrying pTrcHis2A transformed into TOP10 strain
We extracted the vector carrying Moth_1201, 1202, 1204, 2314, 1197, 1198, 1199, 0109, 1191, 1516 from transformed E. coli TOP10 strain.
Results
PCR for TOPO cloning
With the extracted vectors, we performed PCR to amplify each inserted genes to further use them for TOPO vector cloning. As we always do, elongation time was set as 1 min/kb.
Results
With these amplified genes, we performed PCR purification. Concentration and purity are as follows.
Back to the Calendar
Back to the CalendarJune 2nd 2012
PCR amplification of control gene
We PCR amplified a gene to use as a negative control when evaluating the expression result of TOPO cloning.
Results
Control gene is about 800nt long. We purified this gene and its concentration was 216.0 ng/µl with purity of 1.80.
TOPO cloning
With the PCR amplified and purified genes, we performed TOPO cloning by inserting those genes into pBAD TOPO vector to examine the expression of genes with different inducing agent. Ligated TOPO vector was transformed into E. coli TOP10 strain by heat shock transformation method. Transformed cells were spread on LB agar plate for further colony check.
Moth_0109, 1191, 1516 expression check
Cells transformed with pTrcHis2A vectors carrying each of the genes Moth_0109, 1191, 1516 were seeded to examine the expression of each gene. As this vector carries lac operon, genes are induced by treating IPTG of proper amount. Six hours after induction, cell culture will be harvested and the amount of target proteins will be assessed by SDS PAGE. (Both soluble and insoluble fractions of proteins were assessed.)
Back to the Calendar
June 3rd 2012
Colony PCR: 12 transformed genes and control gene
We performed colony PCR with 12 transformed samples and a control to verify if the transformation of cloned TOPO vector into TOP10 strain was successful. Total three colonies were selected from each samples.
Results
Some bands of samples failed to appear in the right place such as Moth_0109 and 2314.
Re-colony PCR: Moth_0109, 2314
Since two samples did not show any successful result in colony PCR and we just gave it another try.
Results
As we can see, Moth_0109 genes amplified through colony PCR has different length compared to the gene construct originally inserted into the vector. This means that Moth_0109 failed to be cloned into the vector and thus transformation was not successful either.
Moth_0109, 1191, 1516 SDS PAGE
With the seeded samples on 6/2, we induced the cell cultures and performed SDS PAGE to see the expression of each gene.
Colony selection and seeding for sequencing
From successfully transformed samples, we selected colonies and cultured them for sequencing. Once their vectors are extracted, they will be sent to Xenotech for sequencing.
Back to the Calendar
June 4th 2012
Colony PCR: Moth_0109 (10 samples)
Since Moth_0109 gene did not show any satisfactory result from colony PCR, we tried it again to figure out the problem.
Results
PCR results showed multiple bands again. Moreover, bands are not in the correct position.
Vector mini-prep for sequencing
We extracted vectors from 11 successfully transformed samples of TOP10.
Results
Among these samples, those whose concentration exceeded 100 ng/µl were sent for sequencing.
Back to the Calendar
June 5th 2012
No special event!
Back to the Calendar
June 6th 2012
No special event!
Back to the Calendar
June 7th 2012
No special event!
Back to the Calendar
June 8th 2012
No special event!
Back to the Calendar
June 9th 2012
No special event!
Back to the Calendar
June 10th 2012
No special event!
Back to the Calendar
June 11th 2012
No special event!
Back to the Calendar
June 12th 2012
No special event!
Back to the Calendar
June 13th 2012
No special event!
Back to the Calendar
June 14th 2012
No special event!
Back to the Calendar
June 15th 2012
No special event!
Back to the Calendar
June 16th 2012
No special event!
Back to the Calendar
June 17th 2012
No special event!
Back to the Calendar
June 18th 2012
No special event!
Back to the Calendar
June 19th 2012
No special event!
Back to the Calendar
June 20th 2012
No special event!
Back to the Calendar
June 21st 2012
No special event!
Back to the Calendar
June 22nd 2012
Moth_0109, 1202 colony PCR
Results
There is no band in the gel. We failed TOPO cloning of Moth 0109, 1202.
Back to the Calendar
June 23rd 2012
Moth_0109, 1202 colony PCR
Results
There is no band in the gel. We failed TOPO cloning of Moth 0109, 1202.
Back to the Calendar
June 24th 2012
Moth_0109, 1202 PCR result
Results
We failed Moth_0109, 1202 TOPO cloning so we tried it again. The PCR band is correct. It means we were successful to make insert DNA.
pBAD TOPO enzyme cut extraction
Results
We didn’t have the TOPO solution anymore. So we tried to reuse the TOPO vector from the other cloned vector. We cut the vector using same restriction enzyme with Moth 0109, 1202 insert and extracted the vector.
Back to the Calendar
June 25th 2012
No special event!
Back to the Calendar
June 26th 2012
No special event!
Back to the Calendar
June 27th 2012
No special event!
Back to the Calendar
June 28th 2012
No special event!
Back to the Calendar
June 29th 2012
No special event!
Back to the Calendar
June 30th 2012
No special event!
Back to the Calendar
