Difference between revisions of "Team:RHIT/Notebook"

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Notebook

What should this page have?

• Chronological notes of what your team is doing.
• Brief descriptions of daily important events.
• Pictures of your progress.
• Mention who participated in what task.

6/8/15-6/12/15

6/11/15

p416-GPD was streaked on LB+Amp plate

6/15/15-6/19/15

6/14/15

Four overnight cultures of p416-GPD colonies were started.

6/15/15

We performed minipreps on overnight cultures of p416 and recovered eight aliquots of 50 µL. Miniprep #s 1, 3, 5, and 7 were digested with EcoR1. When run on a .9% gel, confirmed that minipreps are concentrated enough to use.

6/16/15

IDT constructs 1 and 2 were resuspended to 10 ng/µL.

6/17/15

The iGEM shipping vector, pSB1C3, and construct 2 (the translational unit) were digested with EcoRI and PstI and the resulting fragments were ligated. The NEBuilder procedure was also used to assemble ligated pSB1C3 and construct 2 from IDT. Both NEBuilder assembled, and ligation assembled plasmids were transformed with E. coli and 100 µL aliquots were plated on LB+chlor plates.

6/18/15

Construct 2 and p416 (from miniprep #1) were digested with SpeI and XbaI. Digests were purified using the QIAquick Gel Extraction Kit.

6/19/15

The gel purified fragments from yesterday were concentrated by ethanol precipitation, succeeded by resuspension in TE. V was resuspended in 10 µL and I was resuspended in 40 µL. The entire 10 µL V was ligated with 7 µL I and transformed into E. coli.
More plates of the pSB1C3/T.U. ligation and NEBuilder Assembly were plated (2 plates with 300 µL each).

6/22/15-6/26/15

6/22/15

"No colonies were seen on the p416/T.U. ligation plates, but colonies were seen on pSB1C3 plates from Friday. Eight colonies were picked and grown up in 3 mL cultures overnight.

6/23/15

The rest of the p416/T.U. ligation was plated (350µL on each of 2 plates). Boiling preps were performed on the 3 mL cultures of pSB1C3/T.U.

6/24/15

There are colonies on one of the p416/T.U. plates, so we started 3 mL cultures of 10 colonies (A1-9, B1) for boiling preps tomorrow. Also, boiling preps of pSB1C3/T.U. (from yesterday) were digested with PvuII and SpeI. Bands of 1703 and 847 were expected. After running on a gel, three correct samples were found: NB1 (NEBuilder Assembly, plate B, colony 1), LA3 (Ligation assembly, plate A, colony 3), and LB2 (Ligation assembly, plate B, colony 2).

6/25/15

Fresh 3 mL cultures of three correct samples from yesterday were started. Overnight cultures for A1-8 grew, so boiling preps were performed on these. The preps were then digested with XbaI and PvuII and run on a gel, expecting correct bands at 4450, 879, and 931. It is possible that the insert ligated in the opposite direction due to compatibility between XbaI and SpeI sites. If this happened, we would expect bands at 4450 and 1810 because by mismatching of cohesive ends, the XbaI site is eliminated. On the gel we saw bands at ~1300, ~3000, and ~4500.

6/26/15

Minipreps were performed on NB1, LA3, and LB2 cultures. Minipreps were then digested with SpeI to be run on a gel. Also today, 6 more transformants from p416/T.U. plate B (from 6/23) were used to create a master plate in order to start new cultures.

6/29/15-7/3/15

6/28/15

A gel was run on SpeI digested NB1, LA3, and LB2 to affirm that we have the correct plasmid.

6/29/15

A dot analysis was performed using NB1, LA3, and LB2 to estimate concentrations for sequencing. The resulting estimations were: NB1 - 180 ng/µL; LA3 - 180 ng/µL; LB2 - 100 ng/µL.

6/30/15

Remaining p416/T.U. ligation mixture was used in 6 transformation reactions: three 1 µL reactions (1, 2, and 3), and three 2 µL reactions (4, 5, and 6). Transformations were plated onto twelve plates, two plates (A and B) for each of the six transformations with 200 µL on each plate.

7/1/15

Colonies were found on five of the twelve plates from yesterday, and colonies were picked to start 3 mL overnight cultures (8 from 4A, 2 from 5A, 5 from 5B, 7 from 6A, and 10 from 6B).

7/2/15

Twenty-seven of the thirty-two cultures grew, and boiling preps were performed on these cultures. Then, boiling preps were digested with PvuII and SpeI and run on a gel. All samples returned incorrect results.

7/6/15-7/10/15

This week was taken off as a team break

7/13/15-7/17/15

7/13/15

p416-GPD was streaked on LB+Amp plate

7/14/15

p416-GPD was streaked on LB+Amp plate

7/15/15

p416-GPD was streaked on LB+Amp plate

7/16/15

p416-GPD was streaked on LB+Amp plate

7/17/15

p416-GPD was streaked on LB+Amp plate

7/20/15-7/24/15

7/20/15

p416-GPD was streaked on LB+Amp plate

7/21/15

p416-GPD was streaked on LB+Amp plate

7/22/15

p416-GPD was streaked on LB+Amp plate

7/23/15

p416-GPD was streaked on LB+Amp plate

7/24/15

p416-GPD was streaked on LB+Amp plate

8/3/15-8/7/15

8/10/15-8/14/15

8/17/15-8/21/15