Team:The Agency Escondido/notebook

April 9

Andrew Buss prepared 120 mL of LB broth

April 10

Andrew Buss prepared two LB plates without antibiotics, using our LB plate protocol, rev 72.

Andrew Segina and Vansh Singh inoculated two ampicillin/kanamycin LB plates with E. coli containing Parts A and B from the 3A assembly kit

Andrew Buss inoculated an SOB plate with NEB10 competent E. coli

April 11

Andrew Segina inoculated an SOB plate with NEB10 competent E. coli

The plates previously inoculated with E. coli with parts A and B do not show visible growth

Andrew Segina prepared 1 L of YPD broth without dextrose

April 12

Andrew Buss inspected plates at 8:15 AM. Both NEB10 plates showed visible growth. Neither plate with Part A or B displayed growth.

Andrew Buss prepared four YPD plates from the 1L stock and mixed 4 grams of table sugar before pouring

Andrew Buss inoculated two LB tubes each with E. coli with parts A and B directly from the agar stabs included in the 3A assembly kit. The intention was to diagnose the lack of growth on the A and B plates over two days.

Andrew Buss inspected plates again around 1:00 PM. NEB10 plates showed additional growth, and the plate with Part A contained visible colonies. The plate with Part B did not show growth, however.

Grant Hassinger mixed 100 mL of LB broth and began an autoclave cycle with the media

Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part A plate

Vansh Singh inspected Part B plate at 10:20pm. Part B plate showed visible growth.

Vansh Singh inoculated a tube of ampicillin/kanamycin LB broth from the 3A assembly kit with a colony from the Part B plate

Vansh Singh prepared a tube of ampicillin/kanamycin LB broth from the 3A assembly kit without inoculation. The tube was placed into the refrigerator

Andrew Buss inoculated a tube of LB broth with a colony from the NEB10 plate from April 10

April 13 labeled media

Andrew Segina	LB plate	Part A from kit	Ampicillin, kanamycin
Andrew Segina	LB plate	Part B from kit	Ampicillin, kanamycin
Andrew Buss	SOB plate	NEB 10 from kit
Andrew Segina	SOB plate	NEB 10 from kit
Andrew Buss	LB tube	Part A from kit		Incubating in dry bath
Andrew Buss	LB tube x2	Part A from kit		Incubating in dry bath
Andrew Buss	LB tube x2	Part B from kit		Incubating in dry bath
Vansh Singh	LB tube	Part A from 4/10 plate		Incubating in dry bath
Vansh Singh	LB tube	Part B from 4/10 plate		Incubating in dry bath
Andrew Buss	LB tube	NEB 10 from 4/10 SOB plate		Incubating in dry bath

April 13 available media

Andrew Segina	800 mL YPD	Lacks dextrose
Andrew Buss	4 YPD plates
Andrew Segina	8 LB plates
Andrew Buss	~10mL LB tube	In dry bath for comparison
Andrew Buss	~40mL LB broth
Vansh Singh	~5mL LB broth	Ampicillin, kanamycin
Grant Hassinger	100mL LB broth
Grant Hassinger	100mL LB broth

April 16

Our refrigerated centrifuge arrived, removing the final obstacle to 3A assembly. However, the centrifuge did not consistently power up. This issue was initially ascribed to a faulty fuse.

Vansh Singh performed DNA Miniprep for self grown colonies as per the iGEM 3A Assembly Kit

April 17

Upon closer inspection of the centrifuge, it became evident that it was in fact missing a fuse. When a 20A fuse was installed, the centrifuge powered up normally. Several test runs indicated temperature control in the range of -10 to 30 degrees C, and an effective maximum centrifuge speed of 2500 RPM (despite a dial that can be turned to 6,000 RPM).

Vansh Singh transferred DNA of Part A, Part B, and NEB10 colonies to Ependorfs to find concentration of DNA

Andrew Buss prepared 100 mL of LB broth using revision 14 of our LB broth protocol. However, autoclaving was deferred until a larger batch of material needed autoclaving.

Andrew Buss inoculated two tubes of LB broth with E. coli containing Part A and Part B from the 4/10 plates to provide additional miniprep material for the following day. Due to the scarcity of ampicillin/kanamycin media (antibiotics to be ordered soon), these were cultured without antibiotics.

April 18

The tubes containing Part A and Part B had not displayed visible growth from the night before. However, the tubes inoculated on 4/12 displayed growth.

Andrew Buss extracted plasmid DNA containing Part A and Part B from the cells in the 4/12 tubes using the 3A assembly kit miniprep protocol. RNAse 1 was accidentally omitted from the P1 buffer, so it will need to be introduced before transformation. The two resulting samples were placed in the freezer, labeled "ATB A" and "ATB B". Halfway through the process, the non-refrigerated microcentrifuge was relocated to the interior of our refrigerator, because its operation generates enough heat to risk damaging the DNA samples.

Before restriction can proceed with any of our DNA fragments, the concentration of DNA in the samples must be measured. Our lab does not own a nanodrop machine, so Andrew Segina will use one at a local university to perform the measurement.

When the centrifuge was moved, it did not power up. After the purchase of a replacement fuse, the centrifuge's inconsistent operation was revealed as a consequence of an internally loose power cable. Depending on the orientation of the cable, the centrifuge may not turn on.

Vansh Singh researched GFP and RFP genes for project.

Andrew Buss inoculated three LB tubes and one LB plate with NEB10 cells from the 4/10 plate in preparation for transformation the following day.

April 19

All three NEB10 tubes from 4/18 showed significant growth. The plate from 4/18 showed faint streaks but no isolated colonies.

Quantification of the miniprepped DNA samples from the 18th has been postponed to Monday 4/22. Andrew Buss and Vansh Singh chose to continue with the 3A protocol in the interim, beginning with the restriction of the supplied DNA samples.

Andrew Buss completed restriction digest of all four samples of DNA from the 3A assembly kit: Part A, Part B, pSB1C3 plasmid backbone, and RFP control. However, 22.5 uL of distilled water was pipetted into the RFP restriction reaction, as in the other reactions, rather than the 17.5 uL specifically prescribed for the RFP tube.

Vansh Singh completed Restriction Digest of DNA from kit as per iGEM 3A Assembly kit

Andrew Buss completed ligation of the digests of Parts A and B, plus the plasmid backbone, from the earlier restriction.