IGEM:The Citadel/Notebook/Notes Patrick

11:14am
Removed inoculated conical tubes from yesterday from incubator.

Removed the following plates from yesterday from incubator and placed into 4°C. Agar in some plates was deformed and not completely dry KEPT THE FOLLOWING PLATES (Listed according to quality of gel, not necessarily colony formation): BBa_F1610 [100%] - Completely dry. - Many cells, one large cluster. BBa_J45120 [100%] - Completely dry. - Many cells. Some formation along rim of agar. BBa_R0063 [25%] - Completely dry. - Average colonies. BBa_R0063 [25%] - Completely dry. - Average colonies. BBa_R0040 [25%] - Almost completely dry. - Very few colonies. BBa_F2620 [25%] - Completely dry. - Few colonies, but good ones. BBa_F1610 [50%] - Completely dry. - Normal colonies. BBa_E0240 [50%] - Completely dry. - Very few colonies. Colonies along rim. BBa_I0462 [100%] - Some liquid. Some agar on cover. - Many colonies. BBa_I0462 [50%] - Some liquid. - Normal/average colonies. BBa_I0462 [25%] - Slightly more liquid. Liquid agar on 1/2 of rim circumferance. - Few colonies. Colonies along rim. BBa_F2620 [50%] - Liquid agar on 1/3 of rim. - Average colonies. BBa_J45120 [25%] - Few colonies. Small colonies. Bacterial smear covering 1/12 of plate. BBa_C0160 [25%] - Liquid agar on 1/2 of rim. - Very few colonies, but good ones. BBa_B0015 [25%] - Liquid agar on entire rim. - Average colony formation. DISCARDED THE FOLLOWING PLATES BBa_J45200 [25%] - Too much liquid agar on cover. Unsolidified gel. - No visible colonies. BBa_C0160 [50%] - Too much liquid agar on cover. Unsolidified gel. - Irregular colony formation across unsolidified gel. BBa_B0015 [50%] - Completely unsolidified gel. - Bacteria formation on cover. Irregular colonies. BBa_J45120 [50%] - Too much liquid agar on cover. Unstable gel. - Carpet style formation of bacteria. BBa_J23102 [50%] - Too much liquid agar on cover. Unstable gel. - Bacteria formation on cover. BBa_J45200 [50%] - Too much liquid agar on cover. Unsolidified gel. - No visible colonies. BBa_R0062 [50%] - Too much liquid agar on cover. Unstable gel. - Carpet style formation of bacteria. BBa_R0062 [50%] - Agar dripping onto cover, taking colony formations with it. BBa_E0240 [25%] - Completely dry. Agar slightly unstable. - Would be difficult to isolate from. NOTES Need to replate BBa_J45200 [25%], [50%]. Need to replate BBa_J23102 [25%], [50%]. Need to replate BBa_B0015 [50%]. Need to replate BBa_C0160 [50%]. Need to replate BBa_R0062 [25%], [50%]. Need to replate BBa_J4512 [50%]. Need to replate BBa_E0240 [25%]. In the future: - Do not reheat agar solution in water bath during plate pouring. - Ensure that plates have adequate time to dry completely.

12:45pm
Prepare LB-Miller (Amp) broth for x24 5mL liquid cultures Prepare LB-Miller (Amp) agar 500mL - Therefore, use total 1.24mL Amp (50μL/mL stock)

1:55pm
Plated BBa_I20260 [100%], [50%], [25%] Placed plates into 37°C incubator.

~12:30pm
Streak plated from isolations of the following parts and placed into incubator: BBa_R0063 BBa_I20260

~3:00pm
Poured 33 LB-Miller (Kan) plates and 13 LB-Miller (Amp) plates

4:33pm
Inoculated the following and placed into incubator for miniprep tomorrow morning: BBa_I20260 in 5mL LB Miller (Kan) broth BBa_J45120 in 5mL LB Miller (Amp) broth BBa_I0462 in 5mL LB Miller (Amp) broth

9:45am
Yesterday's plating of BBa_I20260 failed. Replated BBa_I20260 Placed into incubator and 9:59am.

9:30am
Worked on theory for most of morning and afternoon.

2:00pm
Inoculated 80μL of LB-Miller (Amp) broth with following parts and placed into incubator. I13521 J23101 J45120 I0462 I20260 (Cultures will be removed tomorrow morning and added to 40μL of glycerol(50%) and stored at -80°C.)

9:30am
Brian made SOC. Humter prepared LB-Miller (Amp) plates.

5:15pm
Inoculated 10mL of LB-Miller (No antibiotic) broth with parts from 07-14 MIT shipment and placed into incubator. Inoculated 3mL of LB-Miller (no antibiotic) broth with Top 10 E.coli and placed into incubater. (For use in growth measurements tomorrow.)

9:00am
Hunter performed miniprep on cultures that were inoculated with 07-14 MIT shipment parts yesterday. Brian electroporated the following parts: - I746350 - I746351 - I746361 - I746362 - I746360 - I746364 - I746365 Note: "2.49 capacitance this time. Normally 2.50." - I746352 - B0034 (Electroporated with our self-made SOC, as were all parts above.) - B0034 (Electroporated with stock SOC.)

9:22am
Started measuring Top 10 E.coli grwoth pattern. Removed 3mL culture from incubator.

9:44am
Measured OD. OD = 1.53A Made 2 1mL culture dilutions: - x100 Dilution: 1000μL H2O + 10μL culture - x1000 Dilution: 1000μL H2O + 1μL culture

10:04am
Returned culture to incubator.

10:15am
Plated both dilutions and placed into incubator.

10:45am
Measured OD. OD = 1.591A Made 2 1mL culture dilutions: - x100 Dilution: 1000μL H2O + 10μL culture - x1000 Dilution: 1000μL H2O + 1μL culture

11:05am
Returned culture to incubator.

10:13am
Plated both dilutions and placed into incubator.

11:51am
Measured OD. OD = 1.593A Made 2 1mL culture dilutions: - x100 Dilution: 1000μL H2O + 10μL culture - x1000 Dilution: 1000μL H2O + 1μL culture

12:009m
Returned culture to incubator.

12:07pm
Plated both dilutions and placed into incubator.

12:48m
Measured OD. OD = 1.565A Made 2 1mL culture dilutions: - x100 Dilution: 1000μL H2O + 10μL culture - x1000 Dilution: 1000μL H2O + 1μL culture

12:52pm
Returned culture to incubator.

1:04pm
Plated both dilutions and placed into incubator.

1:30pm
Brian and Hunter plated the parts that were electroporated earlier today onto LB-Miller (Amp) and placed into incubator at 1:56pm. - I746350 - I746351 - I746361 - I746362 - I746360 - I746364 - I746365 - I746352 - B0034

9:30am
Brian performed miniprep on the following parts: J23101 J23102 B0034 B0015 E0240 F1616 F2620 J0160 J20260 P1010 K19729 K185004 I746350 I746351 I746352 I746360 I746361 I746362 I746364 I746365

12:12pm
Performed restriction digest using New England Bioworks (NEB) protocol for C1 plasmid backbone and part B0015. (No prior Ethanol Precipitation, DPN1 Digest, or PCR Cleanup was performed on the C1 plasmid backbone prior to the execution of this protocol.) Changes to protocol: 1:00pm When pipetting restriction enzymes may have dipped tip in too far, possibly resulting in too much restriction enzyme and possible "star effect". 1:05pm Ran initial PCR 37°C incubation for 22 minutes rather than normal 15 minutes.

2:25pm
Ligation of the aforementioned C1 plasmid backbone and part B0015 (after completed restriction digest).

3:49pm
PCR amplification of the ligate product of the previous C1 and B0015 ligation. PCR incubation started at 3:49pm. (C1+B0015 ligate are being amplified for use in gel electrophoresis.)

Wednesday, September 09, 2010
Hemocytometer Resources http://en.wikipedia.org/wiki/Hemocytometer http://openwetware.org/wiki/Stevej:Cell_counting/hemocytometer http://openwetware.org/wiki/Cell_counting/hemocytometer http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&item=380089722252&rvr_id=136526929845&crlp=1_263602_263622&UA=WXF%3F&GUID=f16e88d912a0a0aad0c74ee7ffd0acb4&itemid=380089722252&ff4=263602_263622

Monday, September 27, 2010
Qiagen Column Recycling Protocol http://openwetware.org/wiki/Qiagen_Buffers

Saturday, October 16, 2010
Test Wiki