Jackie Tam's notebook

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8/12/09 Tuesday

Microscopy 8/12/09 Tuesday

Today's goal: To see if using a different pipetting method to wash and add chemoattractant changes cell behavior or ability to be tracked.

Cells	 4KW (hM4 stables) med-expression; Med.1, 0.5 CS2, Diff. 8/5/09 (6 day on 8/11/09)
   Count: 1.35x10^6/mL total, 1.15x10^6/mL live, 1.55x10^5/mL dead, 88.5% viability (<--averages of two samples)
Type of microscopy

Open chamber (8-well)

Micrscope settings: 20x objective, brightfield, 10s frames, 15 min, red channel
Chemoattractant solutions: mHBSS+2%BSA, 50nM fMLP, 25nM CNO; chemoattractants diluted in mHBSS+2%BSA
Conditions

   *  (-) fMLP, (-) CNO
   *  (+) fMLP, (-) CNO
   *  (-) fMLP, (+) CNO

3 conditions
Number of experiments per condition: 3
Total number of videos: 9
Notes

  1. 60 min. incubation with 200 ug/mL fibronectin from bovine
  2. See statistical response to 25nM CNO.
  3. Do not see statistical response to 50nM fMLP.
  4. Compared today's (-) fMLP, (-) CNO to that of other dates of microscopy; p values LOW. 
      High basal activity for today's experiments.

Protocol:

  1. Check availability of differntiated HL-60!
  2. Check and turn on the microscope, stage heater (to ~38C), camera and computer.
  3. Prepare 15mL mHBSS 1X+2% BSA (300mg). Let is dissolve COMPLETELY. In the meantime:
  4. Coat 9 open chambers (across two 8-chamber units to save incubation time) with 200 ug/mL fibronectin from bovine. 
     Incubate for 1 hour (60 min.) at room temperature. In the meantime, go to steps 5 and 6. When 1 hour is up, go
     to step 7.
  5. Prepare dilutions of chemoattractant with mHBSS 1X + 2% BSA.
     NOTE: each condition requires 200 uL of chemattractant or mHBSS+2% BSA.
  6. Aliquot enough ion-free PBS (~.5 mL per open chamber) for experiment.
  7. After 1 hour incubation of fibronectin, aspirate all fibronectin from chambers.
  8. Wash 3 times with ion-free PBS, but leave the third wash on chambers until ready to plate cells.
  9. Aliquot enough cells for 1 sample plus a count (10 uL).
 10. Count cells using 1:1 ratio by volume of trypan blue and Invitrogen's Countess.
     If count=1x10^6//mL +/- .2x10^6/mL plate 400 uL
 11. Plate appropriate amount of cells.
 12. Incubate at 37C for 15 min.
 13. Pipette out cells (be clean!).
 14. Wash with 400 uL mHBSS + 2% BSA by tilting the entire unit and pipetting down at a slope. Quickly draw the 
     liquid up again (be clean!).
 15. Add 200 uL of chemoattractant vertically and directly to the center of the chamber.
 16. Place on scope, find focus with about 20 cells/view.
 17. Run movie for 15 min. with 10s frames.

Changes from previous experiments:

  1. Washed at an angle.
  2. Added chemoattractant vertically and in the middle.

Results: Cannot see a statistical response to 50nM fMLP. Can see statistical response to 25nM CNO.

Conclusion: Confused on what to do next/whether we shold drop open chamber microscopy.

8/10/09 Monday

posted ‎‎Aug 11, 2009 7:44 PM‎‎ by Jackie Tam [ updated ‎‎Aug 12, 2009 12:40 AM‎‎ ]

Looked at Microscopy 8/7/09 Friday results with both Iowa and Jason. We haven't been able to see a statistical response to CNO. Troubleshoot microscopy.

Helped Katja pick colonies from TOPO TA clones.

8/7/09 Friday

posted ‎‎Aug 11, 2009 7:44 PM‎‎ by Jackie Tam [ updated ‎‎Aug 12, 2009 12:36 AM‎‎ ]

Microscopy 8/7/09 Friday

Cells

4KD high-expression

Type of microscopy

Open chamber (8-well)

Microscope settings

20x objective, brightfield, 10s frames, 10 min., red channel

Chemoattractant solutions

mHBSS+2%BSA, 50nM fMLP, 15nM CNO, (50nM fMLP + 15nM CNO); chemoattractants diluted in mHBSS+2%BSA

Conditions

   *  (-) fMLP, (-) CNO
   *  (+) fMLP, (-) CNO
   *  (-) fMLP, (+) CNO
   *  (+) fMLP, (+) CNO

4 conditions
Number of experiments per condition: 2
Total number of videos: 8
Notes

  1. First attempt at making set of videos using open chambers.
  2. 60 min. incubation with 200 ug/mL fibronectin from bovine
  3. Room Temp. = 65.5F, Stage Temp. = 38.4C

Analysis:

Program: MATLAB Scripts: CellTracker.m (v1.7), CombineResults.m, GenerateHistograms.m Methods:

  1. Deleted tracks continuous for less than 45 frames (kept those present for 45).

Comments: See a response to fMLP only, but cannot see a response to CNO only. <- Problem because these are hM4D stables!

Conclusion: Troubleshoot microscopy.

8/6/09 Thursday

posted ‎‎Aug 11, 2009 7:44 PM‎‎ by Jackie Tam [ updated ‎‎Aug 12, 2009 12:56 AM‎‎ ]

Did two test samples to check out open well chambers (8-well). Movies looked okay. Will try open chambers.

8/5/09 Wednesday

posted ‎‎Aug 11, 2009 7:43 PM‎‎ by Jackie Tam [ updated ‎‎Aug 12, 2009 12:29 AM‎‎ ]

Morning meeting. See yesterday's post for my powerpoint (attached).

Microscopy 8/5/09 Wednesday

Goal: Observe how 4KW hM4D high-expression stable cells (hM4D gene integrated into genome) respond to 50nM fMLP and 15nM CNO.

Cells	 4KW (hM4 stables) high-expression, 7 day
Type of microscopy

Squeeze chambers

Microscope settings

20X objective, brightfield, 10s frames, 15 min, red channel

Chemoattractant solutions	 mHBSS+2%BSA, 50nM fMLP, 15nM CNO, (50nM fMLP + 15nM CNO); chemoattractants diluted in mHBSS+2%BSA
Conditions

   *  (-) fMLP, (-) CNO
   *  (+) fMLP, (-) CNO
   *  (-) fMLP, (+) CNO
   *  (+) fMLP, (+) CNO

4 conditions
Number of experiments per condition

3

Total videos analyzed

12

Notes

  1.  Microscopy 8/5/09 is a repeat of Microscopy 8/4/09 with blocking.
  2. 70 min. incubation with 200 ug/mL fibronectin.

Data:

8/4/09 Tuesday

posted ‎‎Aug 11, 2009 7:43 PM‎‎ by Jackie Tam [ updated ‎‎Aug 12, 2009 12:26 AM‎‎ ]

Microscopy 8/4/09 Tuesday

Cells	 4KW high-expression
Type of microscopy

squeeze chamber

Microscope settings

20x objective, brightfield, 10s frames, 15 min, red channel

Chemoattractant solutions

mHBSS+2%BSA, 50nM fMLP, 15nM fMLP, (50nM fMLP + 15nM CNO); chemoattractant diluted in mHBSS 1X

Conditions

   *  (-) fMLP, (-) CNO
   *  (+) fMLP, (-) CNO
   *  (-) fMLP, (+) CNO
   *  (+) fMLP, (+) CNO

4 conditions
Number of experiments per condition

3

Total number of videos

12

Notes

  1.  Did not block (did not dilute chemoattractant in mHBSS+2%BSA).
  2.  Repeated whole experiment on 8/5/09. See next day's post.

Analysis:

Program: MATLAB Scripts: CellTracker.m (v1.6), CombineResults.m, GenerateHistograms.m Methods: Deleted dead cells, debris, cells that have no detectable shape change.

Data:

Total Speed - 0 (Grey) vs. 50nM fMLP (Red)

Total Speed - 0 (Grey) vs. 15nM CNO (Red)

Total Speed - 0 (Grey) vs. 50nM fMLP and 15nM CNO (Red)

Net Speed - 0 (Grey) vs. 50nM fMLP (Red)

Net Speed - 0 (Grey) vs. 15nM CNO (Red)

Net Speed - 0 (Grey) vs. 50nM fMLP and 15nM CNO (Red)

Straightness - 0 (Grey) vs. 50nM fMLP (Red)

Straightness - 0 (Grey) vs. 15nM CNO (Red)

Straightness - 0 (Grey) vs. 50nM fMLP and 15nM CNO (Red)

SEE EXCEL SPREADSHEET AND POWERPOINT FOR MORE DATA ANALYSIS AND CONCLUSIONS.

(Edit post) | Attachments: 080409_200fn0fmlp0cno_vs_200fn0fmlp15cno_10bin_netspeed.jpg 080409_200fn0fmlp0cno_vs_200fn0fmlp15cno_10bin_straightness.jpg 080409_200fn0fmlp0cno_vs_200fn0fmlp15cno_10bin_totalspeed.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp0cno_10bin_netspeed.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp0cno_10bin_straightness.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp0cno_10bin_totalspeed.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp0cno_10bin_totalspeed.pdf 080409_200fn0fmlp0cno_vs_200fn50fmlp15cno_10bin_netspeed.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp15cno_10bin_straightness.jpg 080409_200fn0fmlp0cno_vs_200fn50fmlp15cno_10bin_totalspeed.jpg 080409_fn_fmlp_cno_spread.xlsx 080509_WT_HL60_FNandorfMLP_4KW_FNfMLPCNO.pptx

8/3/09 Monday

posted ‎‎Aug 11, 2009 7:42 PM‎‎ by Jackie Tam [ updated ‎‎Aug 11, 2009 10:42 PM‎‎ ]

Analyzed microscopy data from 8/2/09. See yesterday's post for data and conclusions.

Discussed with Ben and Iowa about results. Our thoughts:

   * Manual picking of dead cells/debris is too tedious.
   * Above problem will be worse with transients (cells less happy).
   * Could try open well chambers so we can wash.
   * Try stables with fMLP and CNO in squeeze chambers to see if we can get good data. If good, do same with transients.

7/31/09 Friday

posted ‎‎Aug 11, 2009 5:27 PM‎‎ by Jackie Tam [ updated ‎‎Aug 11, 2009 10:04 PM‎‎ ]

Goal: Observe response of WT HL-60 to fMLP with and without fibronectin in squeeze chambers.

Cells used: JKW (WT HL-60), 6 day differntiated

Details: mHBSS 1X + 2% BSA (fresh), 50nM fMLP in mHBSS 1X + 2% BSA, 125 uL of 200 ug/mL fibronectin from bovine, ion-free PBS (wash x3)

Experiments:

   20x objective, brightfield, 10s frames, 15 min. videos.
   12 total, 3 for each condition.

	 	 	 Condition
1'	 5'	 9'	 (-) fMLP, (-) fibro
2'	 6'	 10'	 (+) fMLP, (-) fibro
3'	 7'	 11'	 (-) fMLP, (+) fibro
4'	 8'	 12'	 (+) fMLP, (+) fibro

Observations: 90 min. incubation for fibronectin instead of 60 min.

Data:

Without fibronectin: Grey/Red = +/- 50nM fMLP respectively

With fibronectin: Grey/Red = +/- 50nM fMLP respectively

Conclusion: There is a bigger difference in response to 50nM fMLP WITH fibronectin. Will use fibronectin in all future microscopy experiments.

| Attachments: 073109_0FN_histogram.jpg 073109_200FN_histogram.jpg 080509_WT_HL60_FNandorfMLP_4KW_FNfMLPCNO.pptx

THIS IS A GREAT NOTEBOOK!!! posted ‎‎Jul 31, 2009 11:10 AM‎‎ by Raquel Gomes

It would be a perfect notebook if you add the results and goals! Please do that! Thanks

7/29/09 Wed.

posted ‎‎Jul 29, 2009 12:39 PM‎‎ by Jackie Tam

Morning meeting. See blog later.

CellASIC experiments w/ Jason.

Microscopy with Iowa.

7/28/09 Tues.

posted ‎‎Jul 28, 2009 5:31 PM‎‎ by Jackie Tam [ updated ‎‎Jul 28, 2009 5:35 PM‎‎ ]

Listened in on my friend Lillian's interview with Jason.

Sent out the final order for some reagents; They're mainly beads, PEG linkers and buffers. A lot of dough being spent here!

Was introduced to the Guava by Jason and Albert.

Miniprep'd ligations for fused parts in pDONR221 and the cultures from yesterday.

Currently running MATLAB for the 4 movies I took yesterday.

7/27/09 Mon.

posted ‎‎Jul 27, 2009 5:13 PM‎‎ by Jackie Tam [ updated ‎‎Jul 28, 2009 5:31 PM‎‎ ]

Microscopy with Iowa.

Exper. #

Sample

1	 NoFN_50fmlp
2	 NoFN_0fmlp
3	 200FN_50fmlp
4	 200FN_0fmlp

Researching micro-oxen reagents with Jason and Saber.

Cultured colonies: hM2 BP, P-Rex BP, TOPO P-Rex, TOPO Tiam-1, TOPO hM2

7/24/09 Fri.

posted ‎‎Jul 24, 2009 7:35 PM‎‎ by Jackie Tam

Minipreps 7/24/09

#	 Sample	 Test digested
1'	 P-Rex BP #1

2'	 P-Rex BP #2	 
3'	 P-Rex BP #3	 
4'	 P-Rex BP #4	 
5'	 LPD Full BP

6'	 Dock2 #1

yes

7'	 Dock 2 #2

yes

8'	 Act A 30-612 #1

yes

9'	 Act A 30-612 #2	 yes
10'	 hM4D QC, BP #1

yes

11'	 hM4D QC, BP #2	 yes

Micro-oxen research. We built theoretical constructs for linking beads to Hl-60. WIll buy reagents soon.

7/23/09 Thurs.

posted ‎‎Jul 23, 2009 7:42 PM‎‎ by Jackie Tam [ updated ‎‎Jul 24, 2009 9:06 AM‎‎ ]

Gel purification of Aar1 digest constructs.

Minipreps for hM2 and Tiam-1.

Picked colonies and cultured Act A long, Dock2, P-rex (4), LPD Full

TOPO TA results - Colonies for P-rex 4, P-Rex 6, P-rex 7, 1 colony for update later

7/22/09 Wed.

posted ‎‎Jul 22, 2009 10:03 PM‎‎ by Jackie Tam [ updated ‎‎Jul 23, 2009 9:45 AM‎‎ ]

Morning meeting. See blog.

Colony PCR Screen for 3 already-fused constructs (7/22/09 Minipreps 2nd set)

   It worked!

Minipreps 7/22/09 1st set:

   22 A, B
   23 A, B
   27 A, B
   30 A, B
   32 A, B
   33 A, B

2nd set:

   Car1-FRB     (5)
   SSF-YFP-Rs1.3-ITSN    (8)
   SSF-YFP-??    (15)

TOPO TA Cloning 7/22/09 AND BP Reactions

   Katja Elongase A-tailed and TOPO'd LPD Full, P-Rex 4, P-Rex 6, P-Rex 7 along with Tiam-1, hM2, hM4D. (7 TOPO TA reactions)
   
1	 Gel extract if more than 1 band shown for PCR products.
2

PCR purify (get rid of oligonucleotides)

Digest with DpnI (destroy template plasmid)

Elongase A-tail

TOPO TA

Transform into DH5alpha

   Katja also BP'd all 7 in parallel.

7/21/09 Tues.

posted ‎‎Jul 21, 2009 1:18 PM‎‎ by Jackie Tam [ updated ‎‎Jul 22, 2009 9:12 AM‎‎ ]

Gel purified PCR product of GC/DMSO Phusion LPD Full. Ready to A-tail and TOPO TA clone.

Miniprep'd hM4D QC (2 cultrues) for Ben. ~75 ng/ul ea.

Troubleshot PCR for P-Rex in pCS2 vector. Most reactions came out nicely.

Prepate for A-tailing/TOPO TA of LPD Full, P-Rex, couple other constructs.

7/20/09 Mon.

posted ‎‎Jul 20, 2009 1:37 PM‎‎ by Jackie Tam [ updated ‎‎Jul 21, 2009 1:18 PM‎‎ ]

PCR LPD X 8 GC/DMSO Phusion is the best of 8.

42 transformations of sequenced BPs(?) for Ben. 3 TOP 10 transformations for Katja's ligations.

7/17/09 Fri.

posted ‎‎Jul 17, 2009 7:33 PM‎‎ by Jackie Tam

Minipreps 7/17/09 for Ben

Ligation reaction for Aynur.

   * Vector = pEGFP-SH2
   * Transformed into DH5alpha

JBEI chalk talk

7/16/09 Thurs.

posted ‎‎Jul 16, 2009 11:26 PM‎‎ by Jackie Tam [ updated ‎‎Jul 20, 2009 2:36 PM‎‎ ]

TOPO TA Cloning (Dock2, ActA)

PCR Done for Aynur

Miniprep ActA 30-612, Dock2

Digest 4 of each with AarI (3 hrs)

Run all Aar1 digest on gel

Run ITSN PCR product on gel Digest 5 PCR products with 1 uL DpnI (2 hrs)

Gel extract (2 Dock2, 3 ActA)

Gel extract ITSN

Gel purify (2 Dock2, 3 ActA)

Gel purify ITSN

TOPO TA'd [already A-tailed]

beta-pix, SSF-YFP (F+R), SSF-YFP F + SSF R Transform PCR/digest into OneShot TOP10. (SDM hM4 1, 2, 3, 4, 5?? - Jackie; Kan x 5)

Transformed into DH5alpha. (TOPO _______ J.T. 7/16/09 w/ 7/14/09 A-tailed products)

A-tailed PCR products (7/14/09)

   Location: Bench freezer, bottom shelf, box, green tape (A-tailed PCRs post gel extraction)
Tube

Sample PCR Product Date

1	 Tuba	 7/13/09	 	 	 
2	 Vav FS + R

7/13/09

3	 Vav FL + R

7/13/09

4	 Dock2	 7/13/09	 	 	 
5	 ITSN	 7/13/09	 	 	 
6	 Sos1	 7/13/09	 	 	 
7	 beta-Pix	 7/13/09

7/16/09 TOPO TA, transformed in DH5alpha

8	 (BC) Rs1.3	 7/13/09	 	 	 
	 	 	 	 	 
9	 (BC) hM4D

7/13/09

10	 (AB) SSF-YFP (F+R)

7/13/09 7/16/09 TOPO TA, transformed in DH5alpha **DON'T NEED

11	 (AB) SSF-YFP F + SSF R

7/13/09 7/16/09 TOPO TA, transformed in DH5alpha **DON'T NEED

12	 ActA 30-612

7/14/09 7/14/09 TOPO TA, transformed in DH5alpha. (1 uL) and (2 uL); 7/15/09 Picked and cultured 4 colonies from each plate (8 cultures); 7/16/09 Mini'd (8 preps), AarI digested (4 preps), gel extracted (3 of 4), gel purified (3 of 4)

13	 Dock2 #1

7/14/09 7/14/09 TOPO TA, transformed in DH5alpha. (1 uL) and (2 uL); 7/15/09 Picked and cultured 4 colonies from each plate (8 cultures); 7/16/09 Mini'd (8 preps), AarI digested (4 of 8), gel extracted (2 of 4), gel purified (2 of 4)

14	 Dock2 #2

7/14/09

15	 Dock2 #3

7/14/09

16	 Dock2 #4

7/14/09

PCR 7/15/09 Products

   Location: Bench freezer, bottom shelf, box, green tape (PCR Products 7/15/09)
Tube	 Sample	 Status
1	 LPD Full

2	 LPD Full

3	 LPD Full	 
4	 LPD Full (best of 6)

5	 LPD Full	 
6	 LPD Full	 
7	 Tuba (failed)

8	 ITSN

7/16/09 Gel purified all

Minipreps 7/16/09 Dock2 x 2 and ActA 30-612 x 2

   Location: 1'-4', 9'-12' in blue iGEM box for unsequenced constructs. Threw away the rest of the mini's (black).
#	 Sample
1'	 Dock2 1 uL TOPO #1
2'	 Dock2 1 uL TOPO #2
3'	 Dock2 1 uL TOPO #3
4'	 Dock2 1 uL TOPO #4
5'	 Dock2 2 uL TOPO #1
6'	 Dock2 2 uL TOPO #2
7'	 Dock2 2 uL TOPO #3
8'	 Dock2 2 uL TOPO #4
9'	 ActA 30-612 1 uL TOPO #1
10'	 ActA 30-612 1 uL TOPO #2
11'	 ActA 30-612 1 uL TOPO #3
12'	 ActA 30-612 1 uL TOPO #4
13'	 ActA 30-612 2 uL TOPO #1
14'	 ActA 30-612 2 uL TOPO #2
15'	 ActA 30-612 2 uL TOPO #3
16'	 ActA 30-612 2 uL TOPO #4

AarI Digest-Gel Purifications 7/16/09

   Notes: (!) Re-numbered
Miniprep Tube

AarI Digest-Gel Purification Tube

7/16/09 1' Dock2 1 uL TOPO #1

Dock2 1 uL TOPO #1

7/16/09 3' Dock2 1 uL TOPO #3

Dock2 1 uL TOPO #2

7/16/09 9' ActA 30-612 1 uL TOPO #1

ActA 30-612 1 uL TOPO #1

7/16/09 10' ActA 30-612 1 uL TOPO #2

ActA 30-612 1 uL TOPO #2

7/16/09 12' ActA 30-612 1 uL TOPO #4

ActA 30-612 1 uL TOPO #3

PCR Product Purifications

   Notes: Thrown away. Already have ITSN.

PCR Products 7/145/09 8. ITSN (used) -> 7/16/09 ITSN PCR-gel purification

7/15/09 Wed.

posted ‎‎Jul 15, 2009 5:15 PM‎‎ by Jackie Tam [ updated ‎‎Jul 16, 2009 11:30 PM‎‎ ]

iGEM Team meeting summary

   Morning meeting with the team. Team Dicty and Team HL-60 traded life stories about our respective parts of the project. In short, Team Dicty has been creating AB-BC-CD fusions and have transformed them into Dicty. AB pieces are mostly catalytic domains and CD pieces are mostly localization domains. BC is still a linker piece of DNA that ensures the cloning of AB and CD in the right orientation in the storage or destination vectors. Oliver and crew hope to observe and analyze in-depth the results of transforming these fusions into Dicty. Would they inhibit chemotaxis and therefore the formation of fruiting bodies in Dicty? How can we track and manipulate PIP2 and PIP3 around the cell? Their main assay is [fluorescent] microscopy.

Picking Colonies from TA TOPO

   Picked and cultured 4 white (positive) colonies from each plate. Dock2 #1, Dock2 #2, ActA 30-612 #1, ActA 30-612 #2. Refer to yesterday's entry: 7/14/09 Tues. For 7/16/09: Miniprep, Aar1 digest, run gel, gel purify.

PCR Reactions 7/15/09

   LPD Full had two faint and smaller-than-expected bands. Will run PCR again in 6 different reaction conditions. See below. Tuba and ITSN failed from Katja's round of PCR with the new primers, so will run 1 reaction each with [my] standard conditions.

PCR #

Sample Annealing Tm. (30 sec) # of Cycles Result (1% agarose gel 7/15) Next Step(s) Status

1	 LPD Full

57C 25 2 very very faint bands, primers Re-dilute LPD Full Prep #1 OR increase amount of DNA added to PCR reaction. Troubleshoot PCR.

2	 LPD Full	 60C	 25	 2 very faint bands, primers

Same as above

3	 LPD Full	 57C	 30	 2 very faint bands, primers

Same as above

4	 LPD Full	 60C	 30	 2 very faint bands; best of 6, primers

Same as above

5	 LPD Full	 57C	 35	 2 very very faint bands, primers

Same as above

6	 LPD Full	 60C	 35	 2 very faint bands, primers

Same as above

7	 Tuba	 55C	 25	 No PCR product, primers

Align primer and prep sequences. Troubleshoot PCR.

8	 ITSN	 55C	 25	 1 faint band (~3550 bp), 1 strong band (~1580 bp)

Compare fragment sizes on gel to expected. Align primer to prep sequences. Gel purify. 7/16/09 Gel purified. ***WHERE

PCR Protocol (Standard) with Phusion

Step	 Description	 Conditions
1	 Initial denaturation

98C, 30s

2	 Denaturing	 98C, 10s
3	 Annealing	 Varies; 55C, 30s
4	 Extending	 72C, 30
5	 GO TO STEP 2 and -->

Varies; 25x, 30x, 35x

6	 Final extention

72, 5 min

7	 Storage	 4C, infinity

Ideas for Troubleshooting PCRs - Raise or lower annealing temperature. - Raise or lower the # of cycles. - Raise or lower the amount of template DNA - Align prep and primer and genbank sequences. Compare. - Re-dilute primers and prep templates. - Use NEW ddH2O

7/14/09 Tues.

posted ‎‎Jul 14, 2009 2:12 PM‎‎ by Jackie Tam [ updated ‎‎Jul 16, 2009 12:21 AM‎‎ ]

Reorganized Primers box with Aynur.

   Learned that some primers are wrong. There may be that the sites for future restriction and ligation into other vectors are not correct. There may also be genes that we want to PCR that have an Aar1 restriction site. To avoid cutting at this Aar1 site in the future (for cloning), we need to not only change a base pair on the primer at the Aar1 site, but design a longer primer that binds at least 8 bases away from the Aar1 site to ensure that Phusion polymerase cannot proofread the "mistake".

- alpha-Pix PCR failed again. We now know the reverse primer may be wrong, but we will try PCR'ing it at more lenient conditions. - PCR Dock2 again. We had higher specifity (now 1 band instead of 2) but lower efficiency. - PCR ActA 30-612 (Prep is low, not sequenced). - PCR LPD Full (Prep is not the best, not sequenced, reverse primer may be wrong (marked by Ben)).

PCR #

Sample Annealing Tm. # of Cycles Results Next Step Status

1	 alpha-Pix	 50C	 25	 failed	 Troubleshoot PCR, align sequences

7/15/09 Give up on alpha-Pix

2	 alpha-Pix	 50C	 35	 failed	 Same as PCR #1

Same as PCR #1

3	 alpha-Pix	 53C	 25	 failed	 Same as PCR #1	 Same as PCR #1
4	 alpha-Pix	 53C	 35	 failed	 Same as PCR #1	 Same as PCR #1
5	 Dock2	 57C	 25	 3 bands (>10 kbp; ~6 kbp, ~4.3 kbp)

Gel purify, polyadenlyation, TA TOPO, transform into DH5alpha 7/14/09 polyadenlyated, in Jackie's yellow box; TA TOPO; Transformed TOPO into DH5alpha - 3 uL DNA + 2 uL DNA on carb x 2;

7/15/09 picked and cultured 4 white colonies from each plate.
6	 Dock2	 57C	 30	 same as PCR #5

Gel purify, polyadenlyation 7/14/09 polyadenlyated, in Jackie's yellow box

7	 Dock2	 59C	 30	 same as PCR #5	 Gel purify, polyadenlyation	 7/14/09 polyadenlyated, in Jackie's yellow box
8	 Dock2	 59C	 35	 same as PCR #5	 Gel purify, polyadenlyation	 7/14/09 polyadenlyated, in Jackie's yellow box
9	 ActA 30-612

55C 25 1 strong band ~1800 bp; good Gel purify, polyadenlyation, TA TOPO, transform into DH5alpha 7/14/09 polyadenlyated, in Jackie's yellow box; TA TOPO; Transformed TOPO into DH5alpha - 3 uL DNA + 2 uL DNA on carb x 2;

7/15/09 picked and cultured 4 white colonies from each plate.
10	 LPD Full

55C 25 2 faint bands (~3 kbp, ~2.4 kbp Troubleshoot PCR 7/15/09 Re-PCR'd - See: 7/15/09 Wed.

Elongase A-tailing Reaction

   About Elongase enzyme: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Nucleic-Acid-Amplification-and-Expression-Profiling/PCR/PCR-Misc/Elongase-Enzyme.html
Buffer B

10 uL

dNTPs (2mM)

1 uL

Elongase	 1 uL
PCR Product

38 uL (All if less)

EB Buffer

38 uL - Vol. PCR Product

Total	 50 uL

TOPO Reaction Edit tomorrow.

7/13/09 Mon.

posted ‎‎Jul 14, 2009 1:59 PM‎‎ by Jackie Tam

Team HL-60 meeting with Ben (morning).

Re-run all 12 PCR reactions in case TOPO or other reactions go wrong.

1	 Tuba
2	 Vav (FS + R)
3	 Vav (FL + R)
4	 Dock2
5	 ITSN
6	 Sos1
7	 alpha-Pix
8	 beta-Pix
9	 BC Rs1.3
10	 BC hM4D
11	 AB SSF-YFP (F + R)
12	 AB SSF-YFP (F + AB SSF, R)

- Ran PCR. - Ran PCR products on gel with cyber green stain (1-7, 10) or ethidium bromide (8, 9, 11-12). - Cut out PCR'd fragment from gels. - Purified DNA from gel using QIAquick gel purification kit. 900 uL QG buffer, no isopropanol, eluted in 30 uL EB buffer.

7/7/09 Tues.

Transforming GPCRs etc. posted ‎‎Jul 7, 2009 5:53 PM‎‎ by Jackie Tam [ updated ‎‎Jul 8, 2009 7:41 PM‎‎ ]

Eric and I did a handful of transformations. This means we have minipreps, NanoDrop-ing, and digesting tomorrow.

List of transformations: GPCRs

GRM2 (mGlu2)

Glu glutamate receptor, metabotropic 2 ? DH5alpha N

GRM4 (mGlu4)

Glu glutamate receptor, metabotropic 4 ? DH5alpha N

V1B

vasopressin vasopressin V1B receptor ? DH5alpha Y

AGTR1 (AT1)

angiotensin II angiotensin II receptor, type 1 ? DH5alpha Y

HTR7A (5HT7) 1 ul

seratonin 5-hydroxytryptamine (serotonin) receptor 7A Amp DH5alpha Y

HTR7A (5HT7) 10 ul

" " Amp DH5alpha Y

HTR2B (5HT2) 1 ul

seratonin 5-hydroxytryptamine (serotonin) receptor 2B Amp DH5alpha Y

HTR2B (5HT2) 10 ul

" " Amp DH5alpha Y

GEFs

Vav (Rac GEF)

5 ng/ul Kan DH5alpha Y, toss

Vav (Rac GEF)

1 ng/ul Kan DH5alpha Y

Actin modulators

ActA (30-612)

29 ng/ul Amp DH5alpha Y

ActA (30-612)

1 ng/ul Amp DH5alpha Y

Other

pmaxGFP

1 ng/ul Kan DH5alpha Y (x 10 for Anyur)

pDONR-221

1 ng/ul Kan TG1 Y (x 10 for Aynur)

7/6/09 Mon.

Yields at last posted ‎‎Jul 6, 2009 10:37 PM‎‎ by Jackie Tam [ updated ‎‎Jul 6, 2009 11:03 PM‎‎ ]

Today, Eric and I had miniprep bootcamp with Aynur! I picked up a lot of tips and tricks like (revised to my liking):

   * Aspirating out the supernatant after spinning down cultures for the first time instead of pouring
   * Waiting for 4 minutes (LESS THAN 5!) to achieve maximum lysing of cells after adding lysis buffer (P2)
   * Watching for a "gooey DNA string" when opening the tubes before adding neutralization buffer (N3)
   * Pipetting in wash buffers PB and PE at the upper rim of the QIAprep columns to bind any DNA left on the rims
   * Washing with buffer PB twice for TG1 cells (TG1 have high endonuclease activity)
   * Discarding the flow through of one buffer PE wash and centrifuging at 13,000 rpm for 1 minute TWO MORES TIMES (for TG1 cells only; to get rid of traces of ethanol)

Thanks, Aynur. I'm much better at miniprep-ing now. :)

We also did a "clean-up" all of our tubes. We separated the good enough (>200 ng/ul) minipreps into 3 boxes: GPCRs - iGEM '09, Actin Modulators - iGEM '09, and GEF Activators - iGEM '09. They are currently stored in the -20C under Aynur's bench.

Before leaving, we transormed DH5alpha cells with some of the GPCRs that we chose back in days of our pre-project, which Ben graciously bought for us to screen, along with some plasmids that we did not get good enough minipreps for. Tomorrow, most of us iGEM-ers have a lecture at 10 on microscopy, and we will do minipreps again.

6/31 - 7/2

If at first you don't succeed, it means you have to do it over and over agian. posted ‎‎Jul 3, 2009 11:35 AM‎‎ by Jackie Tam

More and more transformations, culturing, miniprep-ing, NanoDrop-ing, and sequencing.

6/30 Tues.

More transformations and EZ TaxiScan posted ‎‎Jun 30, 2009 10:19 PM‎‎ by Jackie Tam [ updated ‎‎Jul 6, 2009 10:36 PM‎‎ ]

#	 Plasmid Name

Plate/Resis. Plasmid Code(?)

1	 Beta-pix	 Kan	 MX2-53
2	 Tiam-1	 Carb (Amp)	 MX2-58
3	 Dock2	 Carb (Amp)

MN3-8

4	 Vav	 Kan	 MN1-15
5	 Tuba	 Kan	 MX2-26
6	 Sos1	 Carb (Amp)	 02-9
*7	 Alpha-pix	 Kan	 02-17
8	 Intersectin	 Kan	 Intersectin
9	 P-Rex	 Kan	 R1-26
10	 EGFP-VASP	 Kan	 EGFP-VASP
11	 LPD-Full	 Carb (Amp)	 LPD Full
12	 GST-LPD 775-1250

Carb (Amp) GST-LPD

13	 ActA 30-612

Carb (Amp) ActA 30-612

14	 ActA 255-392

Carb (Amp) ActA 255-392

**15	 B2AR

? B2AR

**16	 DOR-VILLIN	 ?	 DOR-Villin
**17	 Actinin	 ?	 Actinin
**18	 DOR ERM

? DOR-ERM

**19	 DOR EZRIN

? DOR-EZRIN

**20	 DOR KIFC

? DOR-KIFC

**21	 DOR	 ?	 DOR

The plasmid in blue, alpha-pix 02-17, did not grow on the kanamycin plate. Eric and I transformed TG1 cells with alpha-pix 02-17 again. Ben plated the alpha-pix on one kan plate and one carB plate.

- Plasmids in red were plated on unmarked (unknown antibiotic) plates. Eric and I re-did these transformations and plated them on carB (Amp) plates.

Eric and I picked two colonies from each transformation (plasmids 1 through 6, 8 through 14) and cultured the TG1 cells for 5 hours.

Eric and Katja did minipreps for those 26 cultures. Sample digests will determine whether we need to miniprep some of these cells again.

While waiting for them to finish, I visited Arthur and ran through "how to use the EX TaxiScan." I made a video with 5-day differentiated neutrophils (HL-60s) chemoattracting in response to fMLP.

6/29 Mon.

I just plated 28 dishes of bacteria posted ‎‎Jun 29, 2009 11:09 PM‎‎ by Jackie Tam [ updated ‎‎Jun 30, 2009 10:23 PM‎‎ ]

Yum, appetizing. Almost as delicious-looking as these crayon drinks. (See my blog when I next update it (morning, 6/30).)

I don't have the name of the plasmids. Update this later.

6/30 Tues. Update

TG1 - RecA; David says to look up RecA.

#	 Plasmid Name

Plate/Resis. Plasmid Code(?)

1	 Beta-pix	 Kan	 MX2-53
2	 Tiam-1	 carB (Amp)	 MX2-58
3	 Dock2	 carB (Amp)

MN3-8

4	 Vav	 Kan	 MN1-15
5	 Tuba	 Kan	 MX2-26
6	 Sos1	 carB (Amp)	 02-9
7	 Alpha-pix	 Kan	 02-17
8	 Intersectin	 Kan	 Intersectin
9	 P-Rex	 Kan	 R1-26
10	 EGFP-VASP	 Kan	 EGFP-VASP
11	 LPD-Full	 carB (Amp)	 LPD Full
12	 GST-LPD 775-1250

carB (Amp) GST-LPD

13	 ActA 30-612

carB (Amp) ActA 30-612

14	 ActA 255-392

carB (Amp) ActA 255-392

15	 B2AR

? B2AR

16	 DOR-VILLIN	 ?	 DOR-Villin
17	 Actinin	 ?	 Actinin
18	 DOR ERM

? DOR-ERM

19	 DOR EZRIN

? DOR-EZRIN

20	 DOR KIFC

? DOR-KIFC

21	 DOR	 ?	 DOR

The "?" plates were unmarked.

6/24 Wed.

Team Challenge Day 1 and update on digests posted ‎‎Jun 24, 2009 8:13 PM‎‎ by Jackie Tam [ updated ‎‎Jun 24, 2009 8:24 PM‎‎ ]

Black Team! Oliver, Delquin, Saber, Jacinto, Jackie, Alex, Ethan. You guys will rock tomorrow.

My digests are currently in the fridge in a pink box.

From right to left: pHO 43-92 1 through 10

6/23

Minipreps (again) posted ‎‎Jun 23, 2009 3:08 PM‎‎ by Jackie Tam

We're still missing pHO 43-92, 50 and 66 (no positives). We did minipreps for those clones, this time ten cultures for each. We'll do digests later on today, after when ideas are due.

6/19

Digest gels and splitting Dicty cells posted ‎‎Jun 21, 2009 2:22 PM‎‎ by Jackie Tam

We ran our digest gel on a 2 by 16 well 1% agarose gel. We poured our gels on a high-surface tension platform, using two binder clips to prop up two 16-teeth combs. Three pennies propped the platform up against the counter.

Ingredients:

   Pvu1 digest, 10X BPB

Gel photo:

TOP:

Lane	Sample
1	 DNA Ladder
2	 pHO 43-92 1
3	 pHO 43-92 2
4	 pHO 43-92 3
5	 pHO 43-92 4
6	 pHO 43-92 5
7	 pHO 43-132 1
8	 pHO 43-132 2
9	 pHO 43-132 3
10	 pHO 43-132 4
11	 pHO 43-132 5
12	 pHO 43-143 1
13	 pHO 43-143 2
14	 pHO 43-143 3
15	 pHO 43-143 4
16	 pHO 43-143 5

BOTTOM:

Lane	Sample
1	 DNA Ladder
2	 pHO 43-66 1
3	 pHO 43-66 2
4	 pHO 43-66 3
5	 pHO 43-66 4
6	 pHO 43-66 5
7	 pHO 43-99 1
8	 pHO 43-99 2
9	 pHO 43-99 3
10	 pHO 43-99 4
11	 pHO 43-99 5
12	 pHO 43-144 1
13	 pHO 43-144 2
14	 pHO 43-144 3
15	 pHO 43-144 4
16	 pHO 43-144 5

We split our Dicty cells and added fresh media.

Dicty up close and minipreps posted ‎‎Jun 17, 2009 8:53 PM‎‎ by Jackie Tam

When looked at under the microscope, the dicty cells were less than 30% confluent (20-25% maybe? I'm not the best at determining confluency even though it's not that hard. :P). I saw tiny black flecks everywhere in the media. Those were bacteria that survived or were resistant to the antibiotics in the media. We will check the dicty again tomorrow and periodically to monitor confluency. We will split the cells when the confluency is high.

We cultured our cells overnight starting yesterday, so we did minipreps today. Each sample of purified plasmid DNA is now in 50 uL elution buffer in microcentrifuge tubes stored in boxes in the freezer.

Pre-project round 2

posted ‎‎Jun 16, 2009 10:34 PM‎‎ by Jackie Tam [ updated ‎‎Jun 17, 2009 8:43 PM‎‎ ]

Our results weren't what we expected last time, so we're doing mini-preps again. This time, we picked 5 colonies of each clone to culture overnight. We'll work with them tomorrow. We won't fit a team's samples on one 40-lane gel. We'll need lanes for 40 samples plus one for markers!

An encounter with dictyostelium discoideum

posted ‎‎Jun 16, 2009 10:28 PM‎‎ by Jackie Tam [ updated ‎‎Jun 17, 2009 8:43 PM‎‎ ]

We watched the video of dicty cells coming together through AMP chemotaxis again. We got to see real fruit bodies under the microscope, something I don't remember anything about since he introduced dicty to us a month ago. Good review today.

Oliver let us pick some live dicty cells off a plate of media and bacteria to make our own liquid cultures of dicty. We will check on them tomorrow afternoon after bootcamp lectures.

Bootcamp Day 2

posted ‎‎Jun 16, 2009 3:13 PM‎‎ by Raquel Gomes [ updated ‎‎Jun 16, 2009 3:21 PM‎‎ by Jackie Tam ]

Just finished lab safety training. Accidentally editted on Raquel's account!

Jackie Tam's notebook

From 2009.igem.org

Revision as of 07:08, 14 October 2009

Contents

8/12/09 Tuesday

8/10/09 Monday

8/7/09 Friday

8/6/09 Thursday

8/5/09 Wednesday

8/4/09 Tuesday

8/3/09 Monday

7/31/09 Friday

7/29/09 Wed.

7/28/09 Tues.

7/27/09 Mon.

7/24/09 Fri.

7/23/09 Thurs.

7/22/09 Wed.

7/21/09 Tues.

7/20/09 Mon.

7/17/09 Fri.

7/16/09 Thurs.

7/15/09 Wed.

7/14/09 Tues.

7/13/09 Mon.

7/7/09 Tues.

7/6/09 Mon.

6/31 - 7/2

6/30 Tues.

6/29 Mon.

6/24 Wed.

6/23

6/19

Pre-project round 2

An encounter with dictyostelium discoideum

Bootcamp Day 2