Images from yesterday's PCRs
- Gels run for 30 minutes at 9:30 AM
- Same gels run for an additional 15 minutes at 2:00 PM
From the gel with the different controls, we can conclude that either the Vent polymerase or the buffer is contaminated. Also, we think that the biobricks primers are contaminated - we will diagnose this further in the next PCR reaction.
Once again we received plenty of good feedback from our weekly meeting.
- There was a strong sentiment that we should synthesize just the KaiABC genes, instead of synethesizing complete operons including KaiABC and the promoters we want.
- + We can order the syntheses sooner, without having to decide on promoters
- + There's a strong chance we'll want to experiment with different promoters, so our synthesized constructs should be modular
- - However, we could still design our operons to have digestible promoters\
- + We would have BioBrick parts for the registry
Agenda for the next few days
We organized a nice little flowchart going over what we want to do today and tomorrow:
We determined that the Vent or Vent buffer was contaminated with our template, which caused the streaks in our controls. We decided to do another transformation using the second gel-extracted DNA (also from reaction #21 on Monday), which we will then miniprep and PCR with Hotstar and possibly new Vent and/or KOD polymerase.
We incubated 6 colonies from Hetmann's plates in order to increase our chances of getting our 3kb Kai insert. After a lengthy diagnosis, we determined that the 3kb segment we have been working with may not be KaiABC, so this will help us cover our bases.
We transformed OneShot Top10 compentent cells with our KaiABC construct from the second gel purification. This is in preparation of another miniprep and PCR with Hotstar/Vent/KOD.
We used 40 µL, 10 µL, and 10 µL of competent cells for the experimental, positive control, and negative control transformations.
Western Blotting Protocol
Found some resources on Western Blots, which can be found here.
Q: What is the best way to synthesize? With terminator and promoters or without?
Prof. Silver and other faculty believe that synthesis of ORF's is best; while Prof. Church had the idea that they could be made easily swappable out if we could put in BB ends. Still under investigation.
In order of our interest:
WT genes, $1.10/bp if it is not difficult, but if it is difficult it goes to $2.90/bp. 15 days rush delivery included of the 1.6kb construct. Ela is the contact. Sent coding regions for analysis, see peng for email contact.
Cost: $3080 for 15 days guaranteed.
Guaranteeing expression of proteins in e. coli, manipulate codon usage --- guarantees protein expression: $1/bp; pcr, clone; turnaround 7-10days. (will guarantee expression). BUT the catch is that it takes 1 wk to assemble short fragments, 2.5 weeks to assemble long fragments. Dr. Joseph Kiddel is the contact at ext. 1006. For rush assembly of the 1.6kb fragment, it is $3/bp.
Cost: If rush 1.6kb and slow others, $4680+$1200. Otherwise slow is $2800.
$1.50 bp, minimal $500/gene. Turnaround time 1.5kb -> 10-12 business days, guarantee to ship in 20, offer to ship quicker. Clonal in e. coli plasmid. No guarantee of the actual expression. $4/bp for 2-day less than 1kb.
Cost: $4200 for 20 days max, 10-12 avg. Called again, will not drop time guarantee.
Codon devices 4-6 weeks. Pluck 19 vector . Expedited 15day, ADD .30/bp. THEN $1.19/bp ==> $1.49.
Cost: $4172 for 15 days
Blue HeronWon't work