Difference between revisions of "Team:Bilkent-UNAMBG/Notebook"

1st week:

Constructs for IDT order and test constructs with the IDT sequences had planned. Biobricks that we need had ordered. Primers for test constructs had been designed.
 
2nd and 3rd weeks:
Biobricks that we had in part plates had transformed and minipreped.

4th week:
Biobricks ordered had arrived. Except for Q04400, every plate that had been streaked were able to select single colony. For two of the test constructs that we could clone with the DNA we had we had ordered primers for cloning.
I0500 transformed into E. coli dh5a

1st week:
Primers for cloning of two test constructs arrived and were started cloning.
PCR was performed on 5A with S3_T2_2F (57) and K86_T1_R (62) primers in order to create 5B. Annealing temperatures: 57, 60, 62 celcius
K1834847 O/N culture samples were miniprepped.
Gibson assembly was performed with pSB1C3 and 5B and the product (T5) was transformed into E. coli dh5a.
12 colonies from T5 plate were selected and grown O/N.
O/N cultures were miniprepped, digested with HindIII. Unexpected (>10kb) bands were observed. Samples were digested again with NotI and HindIII. 
pSB1C3 linearized plasmid was digested with EcoRI and PstI. Gibson assembly at 1.7.16 was repeated using digested pSB1C3. T5 was transformed into E. coli dh5a.
PCR was performed on K814102 with K86-2_T2_F and K86_T1_R primers in order to produce 2C.
Colony PCR was performed on T5 colonies using S3_T2_SF and K86_T1_R primers. Colonies with positive results were grown O/N.

2nd week:
Test constructs were started clonning 1, 3, 5 and 8. Cloning of test construct 5 was done. In addition, PCR reactions of test constructs 1, 2, 3, 4, 6, and 8 were repeated since they did not work.
O/N cultures were miniprepped and digested with HindIII. Same as before.
EcoRI-PstI digested and not digested pSB1C3 samples were run on gel. Nothing unexpected.
PCR was performed on B0015, S1,R0010, S4A, S2, S7 and S6 in order to produce 1B, 1C, 3C, 8C, 8D, 1A, 3A and 4A. Fail.
PCR was repeated, failed.



3rd week:
PCR reactions of test constructs 2, 3, 4, 6, and 8 were performed for cloning purposes. PCR reactions did not work in spite of optimization for elongation period and TM, even though gradient PCR reactions we couldn’t get any required yield when we ran them on the gel. Induction reactions of decanal sensor was initiated, which is the test construct 5. 
PCR repeat of 1A, 1B,1D,2A3A and 4A with gradient annealing temperature.


4th week:
SDS - PAGE experiment for test construct 5 (decanal sensor) had been performed. No luciferase protein had been detected. PCR 
reactions for test construct 4 had been repeated with different set of primers since the previous reactions were all smear on the
gel. Re-ordered Q04400 had arrived in stabbed agar, inoculated in LB and LB with Kanamycin. T5 containing bacteria induced with 
decanal. O/N culture diluted into OD600 as 1. Half centrifuged and resuspended at PBS. Decanal diluted 1:10, 1:100 and 1:1000 in
water. Decanal added in 99uL cells so that 1%, 0.1%, 0.01% and 0.001% decanal concentration can be obtained. 490nm emission measured
after 2, 10,20 and 30 mins of decanal addition. No difference observed. Possible problem was using clear 96 well plate instead of black.