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From 2012.igem.org

Hyder's lab notes

6/22/12

miniprepped gfp1 and rbs1 and rbs2 liquid cultures

picked 1 colony from double terminator (dt1) plate

picked 1 colony from t7 polymerase (pol1) plate

picked 1 colony from puc19 plate (positive control)

picked 1 colony from dh5a plate (negative control)

started liquid cultures of each colony (5 mL LB amp each)

8/3/12

Resuspended GFPT1 and GFPT2 oligos with molecular grade (nuclease-free) H2O.

Final Concentration 100uM

(gfpt1 top1, gfpt2 top1, gfpt1 top2, gftp2 top2, gfpt1 bot1, gfpt2 bot1, gfpt1 bot2, gfpt2 bot2)

(3uL of each oligo + 2uL 10x annealing buffer, 6uL molecular grade H2O. 20uL Reactions)

Heated for 5 minutes at 100C. Let cool to room temperature on the heating block, stored at -20C.

Digested BBa_I13522 with XbaI and PstI.

Attempted ligating annealed oligos into a digested ?kill plasmid? from Ryan (realized it was cut with E and P).

8/6/12

Annealed oligos for GFPT1 and GFPT2 (target probes)

Ligated oligos with digested GFP plasmid (BBa_I13522)

Transformed into competent DH5alpha

Added SOC and incubated at 37C for 15 minutes.

Plated on amp treated plates.

8/7/12

Only one colony on each plate (both were white)

Picked colonies and started 5mL LB amp cultures of each, stored at 37C

Stored plates in 37C

8/8/12

Picked the colonies again and started new liquid cultures (5mL LB amp).

Discarded cultures for 8/7/12

8/9/12

Miniprepped 3mL of each 8/8/12 culture and nanodropped:

gfpt1 - 155 ng/uL

gfpt2 - 114 ng/uL

Digested gfpt1 and gfpt2 with X and P

Ran on a 1% agarose gel with the digested GFP plasmid

Made glyercol stocks with aliquot of the remaining liquid cultures

8/13/12

Prepared sequencing samples

• Sample w/ Primer*

GFPT1 w/ VF2 GFPT1 w/ VR GFPT2 w/ VF2 GFPT2 w/ VR

200ng of DNA + 16 pmol of primer

Annealed oligos again GFPT1/2

Repeated ligation of oligos with digested GFP plasmid (BBa_I13522)

Followed Haynes assembly protocol instead of standard DH5alpha protocol. (http://openwetware.org/wiki/Haynes:Assembly101)

Transformed ligations into competent DH5alpha

Plated on amp treated plates

8/14/12

Took pictures of plates

Green-white screened plates

Picked 4 white colonies from each of gfpt1/2 plates

Made 5mL LB amp cultures of each colony

Delivered GFPT1/2 dna samples to biodesign for sequencing (samples from 8/13/12)

8/15/12

Miniprepped 3mL of each liquid culture of GFPT1/2

Prepared glycerol stocks using 100uL of each liquid culture

Nanodropped samples:

GFPT1-1 - 172.6 ng/uL GFPT1-2 - 203.7 ng/uL GFPT1-3 - 197.4 ng/uL GFPT1-4 - 178.9 ng/uL GFPT2-1 - 107.3 ng/uL GFPT2-2 - 131.2 ng/uL GFPT2-3 - 145.5 ng/uL GFPT2-4 - 172.0 ng/uL

8/17/12

GFPT1 sequence confirmed

Prepared aliquots of GFPT2 minipreps from 8/15/12 for sequencing

Delivered GFPT2 samples to biodesign for sequencing

8/19/12

GFPT2 sequences confirmed

8/27/12

Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes

Made 4mL cultures in LB Amp

8/29/12

Discarded GFPT1/2 cultures from 8/27/12

Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes

Made 4mL cultures in LB Amp

Digested GFPT1/2 with X and S

Ran a 1% agarose gel with GFPT1/2 digestions

Cut out inserts and GFPT2 backbone and stored in 4C for gel extraction and tandum repeat assembly experiments

8/30/12

Prepared extra glyercol stocks of GFPT1/2 cultures from 8/29/12

Miniprepped 3mL of each culture, stored at -20C

9/19/12

Set up VF2/VR endpoint PCR for double transform minipreps

1-1, 1-1I, 1-2, 1-2I, 1-3, 1-3I, 2-1, 2-1I, 2-2, 2-2I, 2-3, 2-3I, GFPT1 (positive controls), GFPT2 (positive controls)

Annealing temp set to 55C for 25 cycles

Resuspended GFPT1 probe and GFPT2 probe oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C

9/25/12

Resuspended pSB1A2 FWD and pSB1A2 REV (amp resistance primers) oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C

Prepared 1.6uM dilutions (500uL)

Did endpoint PCR using pSB1A2 primer pair on Topo, Topo IPTG, Topo D168A, Topo D168A IPTG, 1-1, 1-1I, 2-1, 2-1I, GFPT1, GFPT2

Did endpoint PCR using VF2/VR primer pair on Topo, Topo IPTG, Topo D168A, Topo D168A IPTG

Annealing temp 55C for 25 cycles, stored products at -20C