Team:Bilkent-UNAMBG/Experiments

Protocols used by BilkentUNAMBG 2016 iGEM Team

Preparation of chemically competent cells (E.coli)

1.We grew a 5mL overnight culture of cells in LB medium in the shaker at 37C.
2.Next day we diluted the culture 1:100 and grew them until it reached an OD600 of 0.2-0.5.
3.The eppendorfs and falcons as well as the TSS buffer were pre-cooled before use.
4.We split the culture into two 50mL falcons and incubated on ice for 10 minutes.
5.They were centrifuged 3000g for 10 minutes at 4C.
6.We removed the supernatant and re-suspended the cells in chilled TSS buffer 10% volume of the amount of cultured centrifuged.
7.100uL aliquots were taken.
8.These were stored in -80C.

Transformation

1.We took the competent cells out of -80C and incubated on ice for 30 minutes.
2.We added 100ng of DNA into the samples and nothing to the controls.
3.We incubated the cells on ice for another 30 minutes.
4.We heat-shocked them at 42C for 30 seconds and incubated on ice for 2 more minutes.
5.We added 500mL LB to the cells and let them recover in the shaker at 37C for 45 minutes.
6.They were then centrifuged at 5800g for 6 minutes.
7.We poured off most of the supernatant and re-suspended the pellet in the remainder.
8.We spread the remainder onto agar plates with the appropriate antibiotics and put them into the incubator at 37C overnight.

MiniPrep of Plasmid DNA (MN Plasmid DNA Purification kit was used during these experiments)

1.We grew bacteria overnight in 10mL LB medium with 1:1000 appropriate antibiotics in 50mL falcons.
2.Next day we centrifuged them at 8000g for 5 minutes and poured off the supernatant.
3.We added A1 buffer and re-suspended the cell pellet by pipetting up and down then transferred the suspension to eppendorfs.
4.We added 250uL A2 buffer and mixed by inverting the tubes a few times. Samples were incubated at room temperature for 5
minutes and no longer.
5.We added 300uL A3 buffer and mixed by inverting the tubes a few times until the blue color of A2 disappeared and the sample
had a consistency similar to cottage cheese.
6.We centrifuged the cells at 13000rpm for 10 minutes.
7.We transferred 700uL from the supernatant to NucleoSpin Columns (as the recommended 750uL usually caused a little overspill
when the cap was closed) and centrifuged at 13000rpm for 1 minute. The flow through was discarded.
8.If there was supernatant remaining, we repeated this step.
9.We washed the columns with 500uL AW buffer and centrifuged at 13000rpm for 1 minute. The flow through
was discarded.
10.We put 600uL A4 buffer on the columns and centrifuged again at 13000rpm for 1 minute. The flow through was discarded.
11.We centrifuged them for an additional 2 minutes at 13000rpm to dry the silica membranes.
12.To elute the DNA, we added 20mL ddH2O -warmed to 65C beforehand- and centrifuged them at 13000rpm for 3 minutes after
waiting for 3 minutes at room temperature.
13.We usually took a second dilution sample by adding another 20uL of ddH2O to the columns and once again centrifuging at
13000rpm for 3 minutes after a 3-minute wait.
14.The concentrations were measured after the process with NanoDrop.
15.The samples were then stored in -20C.

Restriction Enzyme Digestion

1.We followed the NEB protocols for our digestions. We put the recommended amount of restriction enzymes, CutSmart Buffer
and approximately 1000ng of DNA to be cut and completed the reaction volume to 20uL with water.
2.We incubated the samples at 37C for a minimum of two hours.
3.The restriction results were checked by running the samples on agarose gel. If the samples were needed for a following step,
we did gel extraction.

Gel Extraction (MN Plasmid NucleoSpin Gel and PCR Clean-up kit was used during these experiments)

1.After cutting the needed bands out of the agarose gel we weighed them.
2.We added NTI buffer twice the amount of micrograms measured in uL and incubated the samples at 50C 450rpm for 5-10 minutes.
3.We took 700uL form those samples and added on top of NucleoSpin columns. They were centrifuged at 13000rpm for 1 minute then
we discarded the flow through. If there were any remaining solution from the samples we repeated this step.
4.We added 700uL NT3 (with EtOH) and centrifuged at 13000rpm for 1 minute. The flow through was discarded.
5.Step 4 was repeated.
6.We centrifuged the columns at 13000rpm again for 3 minutes to dry.
7.We put the column into new eppendorfs at 65C and incubated approximately 3 minutes.
8.We added 20mL ddH2O -warmed to 65C beforehand- to the columns and centrifuged them at 13000rpm for 3 minutes after waiting
for 3 minutes at room temperature.
9.Step 8 was repeated.

Induction

1.We picked colonies from our plates and put them in 3mL LB medium with 1:1000 appropriate antibiotics. Samples were either
induced with 1:1000 ATC or left uninduced. We also had 1:1000 1M IPTG induced controls. We let them grew in the shaker at 37C.
2.We took periodic measurements of GFP levels at 488nm excitation and 509nm emission with SpectraMax M5 microplate reader.

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