Team:Michigan/Oil Sands October
From 2010.igem.org
(Difference between revisions)
(→10/11/2010) |
(→10/25/2010) |
||
(9 intermediate revisions not shown) | |||
Line 211: | Line 211: | ||
''Marcus'' | ''Marcus'' | ||
- | Overnights made were only 2 mL, and should've been 5 mL. So 3 mL of fresh LB (with antibiotics) was added to 50 mL tubes, and the cultures were added to the fresh LB at 1 pm. Minipreps were made according to the [[Media:MODIFIED_Miniprep_Protocol.pdf|MODIFIED Miniprep]] at room temperature and stored in the | + | Overnights made were only 2 mL, and should've been 5 mL. So 3 mL of fresh LB (with antibiotics) was added to 50 mL tubes, and the cultures were added to the fresh LB at 1 pm. Minipreps were made according to the [[Media:MODIFIED_Miniprep_Protocol.pdf|MODIFIED Miniprep]] at room temperature and stored in the 4ºC. |
- | + | ||
== 10/15/2010 == | == 10/15/2010 == | ||
Line 237: | Line 236: | ||
Incubation: | Incubation: | ||
- | * | + | *37ºC for 15 min |
- | * | + | *80ºC for 20 min |
Ligated according [http://www.roche-applied-science.com/pack-insert/4898117a.pdf Roche Rapid Ligation Protocol]. | Ligated according [http://www.roche-applied-science.com/pack-insert/4898117a.pdf Roche Rapid Ligation Protocol]. | ||
Line 252: | Line 251: | ||
Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]], but made 1:1 and 1:3 dilutions by adding 50 uL of cold, sterile DI water to the comp cells and mixing before each plating. | Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]], but made 1:1 and 1:3 dilutions by adding 50 uL of cold, sterile DI water to the comp cells and mixing before each plating. | ||
- | |||
== 10/16/2010 == | == 10/16/2010 == | ||
Line 263: | Line 261: | ||
Ben started the overnight cultures and assisted in OD measurement and initial centrifugation steps. | Ben started the overnight cultures and assisted in OD measurement and initial centrifugation steps. | ||
- | A colony of P. putida and P. fluorescence was picked from LB agar plates (10/6/10) and grown up in an overnight culture in 2ml Amp at | + | A colony of P. putida and P. fluorescence was picked from LB agar plates (10/6/10) and grown up in an overnight culture in 2ml Amp at 30ºC. |
- | The overnight culture was added to 100 ml of LB media and grown at | + | The overnight culture was added to 100 ml of LB media and grown at 30ºC for 4 hours. |
Protocol was carried out approximately 20 minutes after the following OD600 was recorded: | Protocol was carried out approximately 20 minutes after the following OD600 was recorded: | ||
Line 274: | Line 272: | ||
P. Fluorescence – 0.610 | P. Fluorescence – 0.610 | ||
- | One centrifuge tube containing p. putida culture failed during centrifugation at 5000 rpm. The protocol was altered by reducing the speed to 2500 rpm after this point to reduce the chance of failure. The cells are stored in the | + | One centrifuge tube containing p. putida culture failed during centrifugation at 5000 rpm. The protocol was altered by reducing the speed to 2500 rpm after this point to reduce the chance of failure. The cells are stored in the 4ºC fridge in room 1239. |
== 10/17/2010 == | == 10/17/2010 == | ||
''Marcus and Ben'' | ''Marcus and Ben'' | ||
- | Nanodropped Nick's miniprep of pSB1AT3-Flu (#8) from yesterday, but found low concentrations of about 13-18 ng/uL since each mL of overnight was miniprepped individually. The samples were pooled by using a repurification protocol in the miniprep kit, eluted in Ultrapure water, and recived a final concentration of 40.2 ng/uL. The sequencing core requires 50 ng/uL, so a 5 mL overnight of pSB1AT3-Flu was made for Ben and Nick to miniprep tomorrow and a 2 mL overnight of | + | Nanodropped Nick's miniprep of pSB1AT3-Flu (#8) from yesterday, but found low concentrations of about 13-18 ng/uL since each mL of overnight was miniprepped individually. The samples were pooled by using a repurification protocol in the miniprep kit, eluted in Ultrapure water, and recived a final concentration of 40.2 ng/uL. The sequencing core requires 50 ng/uL, so a 5 mL overnight of pSB1AT3-Flu was made for Ben and Nick to miniprep tomorrow and a 2 mL overnight of DH5α for comp cells. |
== 10/18/2010 == | == 10/18/2010 == | ||
Line 290: | Line 288: | ||
''Marcus'' | ''Marcus'' | ||
- | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from - | + | Made 10 Tet and 10 Cm plates. Also, Nick and I transformed DH5alpha comp cells from -80ºC iGEM box with extra pSB1AT3-J23100-Flu ligation Ben and Kevin made yesterday. Transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. We also transformed ''P. putida'' and ''P. fluorescens'' with pSB1AT3 as a test. No dilutions were made. It was noted while plating the cells that the ''Pseudomonas'' strains somehow wound up with less than 50 uL of cells. And due to the cells flocculating during storage, the ''Pseudomonas'' strains also had be vortexed to resuspend the cells. |
== 10/21/2010 == | == 10/21/2010 == | ||
Line 300: | Line 298: | ||
**VF2- 33.9 nmol/.05 nmol/uL= 678 uL | **VF2- 33.9 nmol/.05 nmol/uL= 678 uL | ||
**VR- 40 nmol/ .05 nmol/uL= 800 uL | **VR- 40 nmol/ .05 nmol/uL= 800 uL | ||
- | *Used a 2 min | + | *Used a 2 min extension |
- | Also, put the Cm and Tet plates from Tuesday in the | + | Also, put the Cm and Tet plates from Tuesday in the 4ºC. |
== 10/23/2010 == | == 10/23/2010 == | ||
Line 312: | Line 310: | ||
'''Expected Digest bands''' | '''Expected Digest bands''' | ||
- | *FimB- 638 bp | + | *FimB - 638 bp |
- | *VP130- 2376, 1925 bp | + | *VP130 - 2376, 1925 bp |
- | *pSB1AT3-Flu- 6770, 15 bp | + | *pSB1AT3-Flu - 6770, 15 bp |
The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | The digests appear to have worked, although the ligations seem to have been incomplete (Roche Rapid Dephos & Ligation Kit). | ||
Line 344: | Line 342: | ||
*16.7 uL UP H2O | *16.7 uL UP H2O | ||
- | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the | + | We then did the following transformations using the [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]] and the DH5α comp cells Nick made using his [[Media:Quick Transformation of E. coli Competant Cell Preperation.pdf| Competent Cell Preparation for Quick Transformation of E. coli]] protocol: |
Transformation (Plate Resistance): | Transformation (Plate Resistance): | ||
Line 372: | Line 370: | ||
Ligation: | Ligation: | ||
- | *pSB1C3- 4.572 fmol/uL | + | *pSB1C3 - 4.572 fmol/uL |
- | *VP130- 8.065 fmol/uL | + | *VP130 - 8.065 fmol/uL |
- | *FimB- 24.2 fmol/uL | + | *FimB - 24.2 fmol/uL |
VP130 Ligation Mix: | VP130 Ligation Mix: | ||
Line 391: | Line 389: | ||
Incubation: | Incubation: | ||
- | *10 mins @ 22. | + | *10 mins @ 22.5ºC |
- | *10 mins @ | + | *10 mins @ 65ºC |
Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. | Ligations were transformed according to [[Media:Direct Plating Transformation Protocol.pdf|Direct Plating Transformation Protocol]]. |
Latest revision as of 01:08, 27 October 2010