Edna Miao's notebook

9/10/09 I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho176 ho180 ho236 ho252 ho269 ho270 ho320 ho335

9/9/09 I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho171 ho173 ho178 ho180 ho181 ho251 ho242 ho239 ho268-ho270 ho240

9/8/09

I froze down ho270 for long term storage.

ho253, 241, 200 got contaminated, and were subsequently tossed out.

9/4/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho245 ho242 ho168 ho230 ho199 ho194 ho266 ho201 ho203 ho268 ho269

9/3/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho159 ho176 ho182 ho207 ho209 ho203 ho227 ho237 ho348

I froze down these strains for long term storage: ho268-269

9/2/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho266 ho267 ho262 ho265

9/1/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho172 ho192 ho196 ho198 ho200 ho197 ho46 ho250 ho249 ho243 ho245 ho248 ho261 ho262 ho264 ho265

Ho260 was contaminated, and ho263 had problems, so we threw both strains and their respective data away.

8/31/09 I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho239-243, 245, 248, 250, 253

I froze down these strains for long term storage: ho266, ho267

8/28/09 I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho239-ho265 ho46

8/27/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho174 ho175 ho168 ho212 ho194 ho211 ho46

I froze down these cells for long term storage: ho239-ho265

8/26/09

I transformed plasmids ho312 and ho313 into HO46 cells.

8/21/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho12 ho351 ho352 ho354 ho356-ho363 ho371 ho372 ho46 ho160 ho194 ho199 ho236 ho230 ho46 from alex

8/20/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho12 ho356-ho363 ho371 ho372

I plated ho12, ho356-363, ho371, ho372 for fruiting bodies.

8/19/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho12 ho351 ho352 ho354

8/17/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho12 ho337-341 ho343 ho344 ho346

I tossed these strains and their subsequent data: ho350 ho353 ho355 ho364 - 370 ho373 because they exhibit phenotype similar to P13K1/2- mutants, an error could have been made in a transformation - transformed into HO2 cells instead of HO12.

8/14/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho46 ho230 ho12 ho337-346 ho310-316 ho321-324

I plated ho46, ho211 for the fruiting bodies assay.

8/13/09

I plated ho12, ho337 - ho346 to check their fruiting bodies

8/12/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: ho12 ho310 - ho316, ho321-ho324

8/10/09

Fruiting Body Assay:

HO46:39 plaques with fruiting bodies.

HO199:3/14 plaques have small fruiting bodies, with one plaque having an abnormally large set of fruiting bodies.

HO 194:0/26 have anything unusual.

HO21:0/29 plaques have anything unusual.

HO211 needs to be redone - only 11 plaques.

HO230:0/30 plaques have anything unusual.

HO235:0/35 plaques have anything unusual.

HO236:0/29 plaques have anything unusual.

HO12:21 plaques have no fruiting bodies.

HO299:72/90 plaques have fruiting bodies.

For more assays, refer to Ethan Chan's notebook.

8/7/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO12, HO300-HO308

8/6/09

Miniprepped 189-44: 133, 60,50, 92, 97, 352, 354 and 189-46: 60, 353, 354

Digested minipreps with PvuI and ran them on a gel. Sequenced 44-354 and 44-353.

Plated PTEN- mutants for fruiting bodies: HO12,HO310-HO319, HO321-HO325

8/5/2009

Froze down HO321-325 for long term storage.

Resequenced 44-353-2.

8/4/2009 I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO12, HO218-HO222 and HO46, HO235-HO236.

I plated these cells for fruiting bodies: HO46, HO235-HO236, HO194, HO199, HO211, HO212, HO230 HO12,HO229, HO237, HO299

I froze down HO310, HO315-HO318 for long term storage.

8/3/09

Fruiting Body Assay HO12: 79/79 plaques have no fruiting bodies

HO218: 34/39 plaques have small fruiting bodies

HO219: 36/40 plaques have regular fruiting bodies HO220: 21/29 plaques have 2 small fruiting bodies and one big one, the rest are regular

HO221: 33/36 plaques have small spread out fruiting bodies

HO222: 39/47   plaques have small fruiting bodies with 3 plaques having large fruiting bodies I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO12, HO218 - HO222 and HO46, HO235, HO236

I Transformed HO221, HO275-HO277, HO279, HO318, HO341, HO360-HO364, HO366, HO367, HO369 into HO12. Duplicates of HO233, HO228, and HO342 were made.

7/30/09

Miniprepped 44-50, 44-60, 44-92, 44-97, 44-352, 44-353, 44-354

Redid Microscopy HO160, 168, 169, 174, 175, 205-207, 211, 212

I froze down HO235-237 for long term storage.

PvuI Digested minipreps

7/28/09

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO205-207, 211, 212.

Fruiting Body Assay Results:

HO207: 0/50 plaques have anything interesting

HO205: 0/32 plaques have anything interesting

HO206: 0/49 plaques have anything interesting

HO46 looked normal as well.

I ligated pHO44 to: pHO92, 97, 50, 60, 352, 353, 354 in storage vector backbone pHO189.

I froze down HO229, HO230 for long term storage.

7/27/09

I plated these strains for Fruiting Body Assay: HO12, HO222, HO220, HO218, HO219, HO221

7/24/09

I plated these strains for Fruiting Body Assay: HO199, HO194, HO212, HO211, HO46.

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO46, HO179, HO199, HO169, HO194, HO175, HO174, HO178

7/23/09

I froze down HO212, HO211 for long term storage

I prepared these cells for microscopy and after filming them, I analyzed their speeds and straightness: HO46, HO199, HO194, HO160, HO179, HO12

I Plated HO207, HO206, HO205 for fruiting body assay.

7/22/09

I transformed pHO235, pHO327, pHO328, pHO306, pHO343, pHO326, pHO329, pHO342, pHO341 into HO46.

I miniprepped 44-133, 44-60, 46-133, 46-60, 46-50.

and used Pvu I to digest the minipreps.

Gel Electrophoresis Sent in positive bands 44-60-2, 44-133-1, 46-133-3 for sequencing.

Microscopy: Took videos of HO46, HO178, HO179. Discarded videos due to incorrect camera settings.

I replated my control plate HO46, because original HO46 had hygromycin added to it and perished.

I froze down HO205-HO207 for long term storage.

7/21/09

I miniprepped 46-50, 44-60, 46-133, 44-133-1, 44-50, 46-50. 444-50-3 got contaminated by centrifuge.

I used PvuI to digest or cut minipreps and then used Gel Electrophoresis and sent in positive bands 44-50-1, 44-50-3, 44-50-4 and 44-60-1 for sequencing.

Microscopy: Took videos of HO169, HO175, HO160, HO168, HO174, HO46, HO179.

Replated HO199, HO194.

7/17/09

Fruiting Body Assay:

HO179: over 15 fruting bodies and their phenotypes are normal

HO168: over 15 fruting bodies and their phenotypes are normal

HO169: over 15 fruting bodies and their phenotypes are normal

HO175: over 15 fruting bodies and their phenotypes are normal

HO174: over 15 fruting bodies and their phenotypes are normal

HO178: over 15 fruting bodies and their phenotypes are normal

7/16/09

I sequenced ALW 148-99-1 and ALW 148-99-3.

I ligated AB piece pHO44 to CD piece pHO50, and transformed ligated construct into DH5alpha cells.

I added hygromycin to yesterday's transformed HO12 cells. I also transformed pHO270, pHO236-242, pHO243-246, pHO318-320, pHO326-329, pHO219-227, pHO229, pHO234, pHO235. The tubes containing pHO223 and pHO232 were mixed up, which resulted in transformed plate pHO223 getting thrown out.

7/15/09

I transformed pHO247, pHO265, pHO266-269, pHO272-274, pHO299-317, and pHO321-324 into HO12 PTEN-, with one control HO12 PTEN-. Grow O/N in HL5, will add hygromycin tomorrow.

Added Hygromycin to yesterday's transformed HO46 cells.

7/14/09

I transformed pHO321, 310,313, 312, 315, 322,324, 307, 308 into Dicty HO46. Grew O/N in HL5 with G418.

I miniprepped PIPK, INP, ALW 148 - 55, ALW 148 - 99, ALW 148 - 66, 44-50, and 46-50.

I cut ALW 148 -99, ALW 148 - 55, ALW 148 - 66, 44-50, and 46-50 with PVU I.

I ran Gel Electrophoresis and only ALW 148-55-2, -3 and ALW 148-99 - 1, -3 were positive. They will be sent in for sequencing when primers are ordered.

7/13/09

I miniprepped storage vectors of ligation of parts: pHO48 - 50,66,92,93,97, pHO46 - 50,93,97 , and pHO44 - 50,97

I cloned AB fragments pHO248, 292, 294 - 298,293 into pHO203.

I transformed pHO318, 319, 299, 305, 323, 306, 275 into Dicty.

7/10/09

Bgl II and Hind III test digests: I cut successful LR reactions of fused parts into destination vectors with Bgl II and Hind III. Ran a gel afterwards to analze fragments, and picked samples for sequencing. For LR reaction data, see Allen Cai or Ethan Chan's notebook, they performed those experiments in my 3 day absence.

7/9/09

I prepared pHO243/HO178 and pHO221/HO168 cells for plating for later fruiting body assays; there was a 1:20 dilution and a 1:100 dilution, for a total of 4 plates.

7/3/09

I ligated AB piece pHO44 with CD pieces pHO50, pHO55, pHO66, pHO92, pHO93, pHO97, pHO99, pHO132, pHO143, pHO144 inside the vector backbone pHO189, with one control that is only pHO189.

7/2/09

I ran Aar1 digests pHO99, pHO44, and pHO132 on a gel, and later purified them,

7/1/09

I cut pHO99, pHO132, pHO44 with Apa 1 to prepare for Aar digest.

I cut pHO203-92, pHO203-93, pHO203-251, pHO203-252, pHO203-259 using Bgl II and HindIII, and incubated at 37*C for 1 hr.

I ran test digests on gel. Bands did not look cut properly, because I used wrong buffer for test digests.

I purified Apa 1 digests: pHO44, pHO99, pHO132.

I used Aar1 to cut pHO44, pHO99, pHO132.

I sent in 203-93-1, 203-92-1, 203-251-1, -2, 203-252-1-2, 203-259-1 for sequencing.

6/30/09

I picked 10 colonies for pHO43-66, and miniprepped them.

Pvu I Test Digest: I cut pHO 43-66. I ran them on a gel. I sent in 43-66-1, -2, -3, -6 for sequencing, and only 43-66-1 worked.

6/29/09

I performed Gateway LR reaction on 14 pHO constructs: pHO248, pHO249, pHO250, pHO251,pHO259, pHO252, pHO253, pHO258,pHO254, pHO255, pHO256, pHO257, pHO92, pHO93 into destination vector pHO203 (Cterm RFP). I transformed them into DH5alpha and grew them O/N on carb plates.

I transformed pHO 44 into DH5 alpha, and grew them O/N on kanamycin plate.

6/25/09

I ran my PvuI sample digests today 145 volts on a 1.0&% agarose gel to see if I had any postives: positives meaning a fragment of DNA that is greater than 3000 bp. This means that that plasmid had worked, and there are vector inserts to that vector backbone.

6/23/09

I cut my 45-55 plasmids with PvuI.

6/23/09

I purified plasmid 45-55 ten times

6/22/09

After redoing our minipreps for plasmids at UCSF, there are still a few plasmids that didn't work. For my team, those plasmids are plasmids 45-50, 45-55, and 45-132. Alex took 45-50, Allen took 45-132, and I was left with 45-132. So today, I prepared 10 minipreps for 45-132. The bacteria are being grown O/N in 5ml of LB broth.

6/19/09

We ran all of our sample digests on a 1% agarose gel today. Of the 15 samples, 6 of mine were clear positives. In other words, plasmids 45-99 and 45-66 were sent in for sequencing. Purification of 45-143 via minipreps will be repeated.

6/18/09

Performed 15 PvuI mini digests today in PCR tubes. We digested purified plasmids from the miniprep done yesterday.

6/17/09 Miniprep Day 2

The plasmids were purified today, and are stored in the fridge.

6/16/09 Minipreps Day One

iGEM pre-project: we're redoing our minipreps due to wack gel results. I had plasmids pho45-66, pho45-99, and pho 45-143 - we cultured bacteria in culture tubes from five colonies from each plate of the respective plasmids - for a total of 15 culture tubes in total. The bacteria will be grown O/N for tomorrow's miniprep.

6/16/09

Today, we split into teams of two, and learned how to plate Dicty from Oliver. Ryan and I removed some cells from a plate of Dicty and transferred it to a separate plate with media. We then placed them into an incubator.