Difference between revisions of "Production/Competent Cells"

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__NOTOC__
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==Batch Summary==
 
==Batch Summary==
 
Use this page to keep track of competent cell production. See the [[Help:Protocols/Competent_Cells | competent cell protocol]] for more information.
 
Use this page to keep track of competent cell production. See the [[Help:Protocols/Competent_Cells | competent cell protocol]] for more information.
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<table >
 
<table >
 
<!-- Table Header (Titles)--!>
 
<!-- Table Header (Titles)--!>
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<tr><th>Batch <th>Strain <th>Maker <th>Date Started <th>Temp, RPM <th>Incubation (hr) <th>Culture Volume <th>Culture OD (600nm) <th>Competent OD (600nm) <th>Cell Count (AVG) <th>Efficiency <th>250ul Aliquots <th>500ul Aliquots <th>1ml Aliquots <th>96w Plate</tr>
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<tr><th>Batch <th>Strain <th>Maker <th>Date Started <th>Temp, RPM <th>Incubation (hr) <th>Culture Volume <th>Culture OD (600nm) <th>Competent OD (600nm) <th>Cell Count (AVG) <th>Efficiency <th>250ul Aliquots <th>500ul Aliquots <th>1ml Aliquots <th>96w Plate <th>Checklist pdf</tr>
  
 
<!-- Expected Values --!>
 
<!-- Expected Values --!>
 
<tr style="background-color:#C0C0C0;"><td>Expected Values<td><td><td><td>20C<td>16<td><td>0.3 or lower
 
<tr style="background-color:#C0C0C0;"><td>Expected Values<td><td><td><td>20C<td>16<td><td>0.3 or lower
−
<td>1 - 1.5<td>100 - 400<td>5*10^8 - 5*10^9<td><td><td><td></tr>
+
<td>1 - 1.5<td>100 - 400<td>5*10^8 - 5*10^9<td><td><td><td><td></tr>
  
 
<!-- Batch 001 Data Starts--!>
 
<!-- Batch 001 Data Starts--!>
−
<tr><td>001 <td>NEB 10b <td>VS+KN <td>8/17/11 <td>21C, 250rpm <td>16 <td>500ml <td>0.4 <td>1.35 <td>67,88,112 (89) <td>1.335*10^8 <td>20 <td>20 <td>10 <td>0</tr>
+
<tr><td>001 <td>NEB 10b <td>VS+KN <td>8/17/11 <td>21C, 250rpm <td>16 <td>500ml <td>0.4 <td>1.35 <td>67,88,112 (89) <td>1.335*10^8 <td>20 <td>20 <td>10 <td>0 <td>NA</tr>
 
<!-- Batch 001 Data Ends--!>
 
<!-- Batch 001 Data Ends--!>
  
 
<!-- Batch 002 Data Starts--!>
 
<!-- Batch 002 Data Starts--!>
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<tr><td>002 <td>NEB 10b <td>VS+KN <td>9/12/11 <td>20C, 250rpm <td>16 <td>500ml <td>0.40 <td> 1.12 <td> ??, ??, ?? (??) <td>?? <td> ?? <td> 0 <td> 0 <td>4</tr>
+
<tr><td>002 <td>NEB 10b <td>VS+KN <td>9/12/11 <td>20C, 240rpm <td>16 <td>500ml <td>0.40 <td> 1.12 <td> 91, 140, 131, 45, 66, 30 (83.3) <td>1.258*10^8 <td> 3 <td> 0 <td> 0 <td>4 <td>Yes</tr>
 
<!-- Batch 002 Data Ends--!>
 
<!-- Batch 002 Data Ends--!>
  
 
<!-- Batch 003 Data Starts--!>
 
<!-- Batch 003 Data Starts--!>
−
<tr><td>003 <td>NEB 10b <td>VS+KN <td>9/12/11 <td>20C, 250rpm <td>16 <td>500ml <td>0.23 <td> 0.71 <td> ??, ??, ?? (??) <td>?? <td> ?? <td> 0 <td> 0 <td>4</tr>
+
<tr><td>003 <td>NEB 10b <td>VS+KN <td>9/12/11 <td>20C, 240rpm <td>16 <td>500ml <td>0.23 <td> 0.71 <td> 127, 145, 72, 83, 93, 60 (96.7) <td>1.450*10^8 <td> 7 <td> 0 <td> 0 <td>4 <td>Yes</tr>
 
<!-- Batch 003 Data Ends--!>
 
<!-- Batch 003 Data Ends--!>
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<!-- Batch 004 Data Starts--!>
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<tr style="background-color:#E0E0E0"><td>004 <td>NEB 10b (002)<td> KN+VS <td>9/18/11 <td>20C, 240rpm <td> 18 <td>500ml <td>0.13<td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA </tr>
 +
<!-- Batch 004 Data Ends--!>
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 +
<!-- Batch 005 Data Starts--!>
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<tr style="background-color:#E0E0E0"><td>005 <td>NEB 10b (002)<td> KN+VS <td>9/18/11 <td>20C, 240rpm <td> 18 <td>500ml <td>0.14<td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA </tr>
 +
<!-- Batch 005 Data Ends--!>
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<!-- Batch 006 Data Starts--!>
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<tr><td> 006 <td>NEB 10b (002)<td> KN+VS <td>9/19/11 <td>20C, 240rpm <td> 21 <td>500ml <td>0.28<td> 0.74 <td> 20, 29, 17, 10, 10, 12 (16.3) <td> 2.45*10^7 <td> 4 <td> 0 <td> 0 <td> 4 <td> Yes </tr>
 +
<!-- Batch 006 Data Ends--!>
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 +
<!-- Batch 007 Data Starts--!>
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<tr><td> 007 <td>NEB 10b (002)<td> KN+VS <td>9/19/11 <td>20C, 240rpm <td> 20 <td>500ml <td>0.23<td> 0.76 <td> 14, 10, 15, 9, 9, 13 (11.6) <td> 1.750*10^7 <td> 7 <td> 0 <td> 0 <td> 4 <td> Yes </tr>
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<!-- Batch 007 Data Ends--!>
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 +
<!-- Batch 008 Data Starts--!>
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<tr><td> 008 <td>NEB 10b (002)<td> KN+VS <td>9/22/11 <td>20C, 240rpm <td> 21 <td>500ml <td>0.27<td> 0.68 <td> TBD <td> TBD <td> 8 <td> 0 <td> 0 <td> 4 <td> Yes </tr>
 +
<!-- Batch 008 Data Ends--!>
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<!-- Batch 009 Data Starts--!>
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<tr style="background-color:#E0E0E0"><td> 009 <td>NEB 10b (002)<td> KN+VS <td>9/22/11 <td>20C, 240rpm <td> 22 <td>500ml <td>0.16<td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA <td> NA </tr>
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<!-- Batch 009 Data Ends--!>
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<!-- Batch 010 Data Starts--!>
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<tr><td> 010 <td>NEB 10b (002)<td> VS+KN <td>9/27/11 <td>20C, 240rpm <td> 22 <td>500ml <td>0.38<td>1.52<td>88, 74, 81, 40, 47, 62 (65.3) <td> 9.800*10^7 <td> 2 <td> 0 <td> 0<td> 5 <td> Yes </tr>
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<!-- Batch 010 Data Ends--!>
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<!-- Batch 011 Data Starts--!>
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<tr><td> 011 <td>NEB 10b (002)<td> KN+VS <td>9/27/11 <td>20C, 240rpm <td> 22 <td>500ml <td>0.46<td>1.47<td> 24, 41, 48, 20, 22, 32 (31.2) <td> 4.675*10^7 <td>8<td> 0 <td> 0 <td> 4 <td> Yes </tr>
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<!-- Batch 011 Data Ends--!>
  
 
</table>
 
</table>
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The efficiency test/test transformation used a single 250ul aliquot (randomly chosen). Three transformations were done using our standard template and protocol. '''Important:''' In following productions we will test the first and last of the aliquots.
 
The efficiency test/test transformation used a single 250ul aliquot (randomly chosen). Three transformations were done using our standard template and protocol. '''Important:''' In following productions we will test the first and last of the aliquots.
 +
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'''Notes:''' While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.
  
 
===002===
 
===002===
 
OD600nm value at 15.5 hours was 0.26, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.40.
 
OD600nm value at 15.5 hours was 0.26, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.40.
 +
 +
'''Notes:''' While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.
  
 
===003===
 
===003===
 
OD600nm value at 15.5 hours was 0.16, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.23.
 
OD600nm value at 15.5 hours was 0.16, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.23.
 +
 +
'''Notes:''' While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.
 +
 +
===004===
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First comp cell batch to use seed stock batch #002. Cells grew very slowly; low OD even after 18 hours of culture. Batch was discarded.
 +
 +
===005===
 +
Cells grew very slowly; low OD even after 18 hours of culture. Batch was discarded.
 +
 +
===006===
 +
Media was inoculated at 4pm on Day 1 to allow for longer growth period. Cells grown in 1L flask. Cells were OD'ed multiple times: 16 hours (0.12), 16.5 hours (0.12), 18.5 hours (0.15), and 21 hours (0.28). Test transformations were performed with the first and last 250ul aliquot.
 +
 +
===007===
 +
Media was inoculated at 4pm on Day 1 to allow for longer growth period. Cells grown in 1L flask. Cells were OD'ed multiple times: 16 hours (0.13), 16.5 hours (0.14), 18.5 hours (0.22), and 20 hours (0.23). Test transformations were performed with the first and last 250ul aliquot.
 +
 +
===008===
 +
Cells grew slowly; temperature was increased to 23C at 19 hours. Cells were OD'ed multiple times: 18 hours (0.09), 19 hours (0.11), and 21 hours (0.27). Final ice incubation step was approximately 25 minutes.
 +
 +
===009===
 +
Cells grew very slowly; temperature was increased to 23C at 19 hours. Cells were OD'ed multiple times: 18 hours (0.04), 19 hours (0.06), 21 hours (0.13), and 22 hours (0.16). Batch was discarded.
 +
 +
===010===
 +
Minimal dry ice available. "Last" 250ul aliquot was aliquoted after finishing 4 plates.
 +
 +
===011===
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Minimal dry ice available. "Last" 250ul aliquot was second to last 250ul aliquot.

Latest revision as of 20:20, 20 October 2011


Batch Summary

Use this page to keep track of competent cell production. See the competent cell protocol for more information.

Batch Strain Maker Date Started Temp, RPM Incubation (hr) Culture Volume Culture OD (600nm) Competent OD (600nm) Cell Count (AVG) Efficiency 250ul Aliquots 500ul Aliquots 1ml Aliquots 96w Plate Checklist pdf
Expected Values20C160.3 or lower 1 - 1.5100 - 4005*10^8 - 5*10^9
001 NEB 10b VS+KN 8/17/11 21C, 250rpm 16 500ml 0.4 1.35 67,88,112 (89) 1.335*10^8 20 20 10 0 NA
002 NEB 10b VS+KN 9/12/11 20C, 240rpm 16 500ml 0.40 1.12 91, 140, 131, 45, 66, 30 (83.3) 1.258*10^8 3 0 0 4 Yes
003 NEB 10b VS+KN 9/12/11 20C, 240rpm 16 500ml 0.23 0.71 127, 145, 72, 83, 93, 60 (96.7) 1.450*10^8 7 0 0 4 Yes
004 NEB 10b (002) KN+VS 9/18/11 20C, 240rpm 18 500ml 0.13 NA NA NA NA NA NA NA NA
005 NEB 10b (002) KN+VS 9/18/11 20C, 240rpm 18 500ml 0.14 NA NA NA NA NA NA NA NA
006 NEB 10b (002) KN+VS 9/19/11 20C, 240rpm 21 500ml 0.28 0.74 20, 29, 17, 10, 10, 12 (16.3) 2.45*10^7 4 0 0 4 Yes
007 NEB 10b (002) KN+VS 9/19/11 20C, 240rpm 20 500ml 0.23 0.76 14, 10, 15, 9, 9, 13 (11.6) 1.750*10^7 7 0 0 4 Yes
008 NEB 10b (002) KN+VS 9/22/11 20C, 240rpm 21 500ml 0.27 0.68 TBD TBD 8 0 0 4 Yes
009 NEB 10b (002) KN+VS 9/22/11 20C, 240rpm 22 500ml 0.16 NA NA NA NA NA NA NA NA
010 NEB 10b (002) VS+KN 9/27/11 20C, 240rpm 22 500ml 0.381.5288, 74, 81, 40, 47, 62 (65.3) 9.800*10^7 2 0 0 5 Yes
011 NEB 10b (002) KN+VS 9/27/11 20C, 240rpm 22 500ml 0.461.47 24, 41, 48, 20, 22, 32 (31.2) 4.675*10^7 8 0 0 4 Yes

Batch Notes

001

This is the first batch attempting to fully standardize competent cell production. Also Kitwa's first batch! And a resounding success!

The efficiency test/test transformation used a single 250ul aliquot (randomly chosen). Three transformations were done using our standard template and protocol. Important: In following productions we will test the first and last of the aliquots.

Notes: While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.

002

OD600nm value at 15.5 hours was 0.26, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.40.

Notes: While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.

003

OD600nm value at 15.5 hours was 0.16, and cells were re-placed in the shaker for another 0.5 hours. OD value afterward was 0.23.

Notes: While ODing culture, cells were not kept on ice/stored in fridge. Poured into centrifuge tubes on ice, but tubes were not pre-chilled.

004

First comp cell batch to use seed stock batch #002. Cells grew very slowly; low OD even after 18 hours of culture. Batch was discarded.

005

Cells grew very slowly; low OD even after 18 hours of culture. Batch was discarded.

006

Media was inoculated at 4pm on Day 1 to allow for longer growth period. Cells grown in 1L flask. Cells were OD'ed multiple times: 16 hours (0.12), 16.5 hours (0.12), 18.5 hours (0.15), and 21 hours (0.28). Test transformations were performed with the first and last 250ul aliquot.

007

Media was inoculated at 4pm on Day 1 to allow for longer growth period. Cells grown in 1L flask. Cells were OD'ed multiple times: 16 hours (0.13), 16.5 hours (0.14), 18.5 hours (0.22), and 20 hours (0.23). Test transformations were performed with the first and last 250ul aliquot.

008

Cells grew slowly; temperature was increased to 23C at 19 hours. Cells were OD'ed multiple times: 18 hours (0.09), 19 hours (0.11), and 21 hours (0.27). Final ice incubation step was approximately 25 minutes.

009

Cells grew very slowly; temperature was increased to 23C at 19 hours. Cells were OD'ed multiple times: 18 hours (0.04), 19 hours (0.06), 21 hours (0.13), and 22 hours (0.16). Batch was discarded.

010

Minimal dry ice available. "Last" 250ul aliquot was aliquoted after finishing 4 plates.

011

Minimal dry ice available. "Last" 250ul aliquot was second to last 250ul aliquot.