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Team:Arizona State/Notebook/hyder
From 2012.igem.org
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Annealing temp 55C for 25 cycles, stored products at -20C | Annealing temp 55C for 25 cycles, stored products at -20C | ||
| + | |||
| + | Made 3mL liquid cultures of the shipping vector (pSB1C3 with RFP insert) in DH5alpha in chloramphenicol resistant LB | ||
| + | |||
| + | Made 10mL liquid cultures of: | ||
| + | Topo in kanamycin | ||
| + | topo D168A in kanamycin | ||
| + | topo + GFPT1 in kanamycin + ampicillin | ||
| + | topo + GFPT2 in kanamycin + ampicillin | ||
| + | |||
| + | stored @ 37C | ||
| + | |||
| + | 9/26/12 | ||
| + | |||
| + | Picked two new colonies for topo + GFPT2 and grew 10mL cultures of amp+kan LB broth for each | ||
| + | |||
| + | Ran a gel with: | ||
| + | Hyperladder I, GFPT1 pSB1A2, GFPT2 pSB1A2, Topo pSB1A2, Topo D168A | ||
Revision as of 10:34, 1 October 2012
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
Made 3mL liquid cultures of the shipping vector (pSB1C3 with RFP insert) in DH5alpha in chloramphenicol resistant LB
Made 10mL liquid cultures of: Topo in kanamycin topo D168A in kanamycin topo + GFPT1 in kanamycin + ampicillin topo + GFPT2 in kanamycin + ampicillin
stored @ 37C
9/26/12
Picked two new colonies for topo + GFPT2 and grew 10mL cultures of amp+kan LB broth for each
Ran a gel with: Hyperladder I, GFPT1 pSB1A2, GFPT2 pSB1A2, Topo pSB1A2, Topo D168A
