Difference between revisions of "Team:Paris Saclay/Notebook/August/4"
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+ | {{Team:Paris_Saclay/notebook_header}} | ||
=Tuesday 4th August= | =Tuesday 4th August= | ||
==Lab Work== | ==Lab Work== | ||
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''by Pauline'' | ''by Pauline'' | ||
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* BBa_K1707008 #1 to #6 | * BBa_K1707008 #1 to #6 | ||
* BBa_I13600 #1 and #2 | * BBa_I13600 #1 and #2 | ||
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''by Pauline and Coralie'' | ''by Pauline and Coralie'' | ||
− | + | * BBa_K1707008 #1 to #6 | |
Mix: | Mix: | ||
Line 30: | Line 30: | ||
Agarose gel 1% | Agarose gel 1% | ||
Migration 90V | Migration 90V | ||
− | + | [[File:ParisSaclay 04.08.15-Digestion vérif 008.jpg|300px|center]] | |
+ | <html><i><p>Verification by digestion with XbaI and PstI of BBa_K1707008, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty</p></i></html> | ||
We can conclude that all BBa_K1707008 clones are OK | We can conclude that all BBa_K1707008 clones are OK | ||
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Migration 100V | Migration 100V | ||
− | + | [[File:Paris Saclay-03.08.2015 - digestion verification 2.jpg|400px|center]] | |
+ | <html> | ||
+ | <i>Verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707021#1, 3. BBa_K1707021#2, 4. BBa_K1707021#3, 5. BBa_K1707021#4, 6. BBa_K1707021#5, 7. BBa_K1707021#6, 8. BBa_K1707011#4, 9. BBa_K1707011#5, 10. BBa_K1707011#6</i></html> | ||
+ | [[File:Paris Saclay-03.08.15 - digestion verification 1.jpg|400px|center]] | ||
+ | <html> | ||
+ | <p><i>Verification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707004#3, 5. BBa_K1707004#4, 6. BBa_K1707004#5, 7. BBa_K1707004#6, 8. BBa_K1707011#1, 9. BBa_K1707011#2, 10. BBa_K1707011#3, 11. Empty, 12. Empty</i></p></html> | ||
We can conclude that: | We can conclude that: | ||
* BBa_K1707021: #2, #3, #4 and #6 are OK | * BBa_K1707021: #2, #3, #4 and #6 are OK | ||
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EcoRI + SpeI: | EcoRI + SpeI: | ||
* BBa_K1707004 | * BBa_K1707004 | ||
− | |||
Mix: | Mix: | ||
Line 79: | Line 84: | ||
* 6 µL H2O | * 6 µL H2O | ||
* 10 µL Plasmide | * 10 µL Plasmide | ||
− | |||
===Purification agarose gel=== | ===Purification agarose gel=== | ||
''by Coralie and Audrey'' | ''by Coralie and Audrey'' | ||
− | |||
* BBa_K1707011 | * BBa_K1707011 | ||
* BBa_K1707004 | * BBa_K1707004 | ||
Line 95: | Line 98: | ||
Migration 100 V | Migration 100 V | ||
− | + | [[File:ParisSaclay 04.08.15-Purification.jpg|400px|center]] | |
+ | <html> | ||
+ | <p><i>Verification of gel purification, from left to right: 1. <a href="https://2015.igem.org/File:Paris_Saclay-Ladder.jpg" target="_blank">DNA Ladder</a>, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty</i></p></html> | ||
We can conclude that I13602 isn't digested | We can conclude that I13602 isn't digested | ||
− | |||
===Purification=== | ===Purification=== | ||
''by Pauline'' | ''by Pauline'' | ||
− | |||
* BBa_K1707011 | * BBa_K1707011 | ||
* BBa_K1707004 | * BBa_K1707004 | ||
Line 118: | Line 121: | ||
===Soil experiment=== | ===Soil experiment=== | ||
''by Coralie'' | ''by Coralie'' | ||
+ | |||
====Water==== | ====Water==== | ||
Sea Water separate in 5mL in 50mL Falcon | Sea Water separate in 5mL in 50mL Falcon | ||
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Water + concentrate cells solution 10 fold dilution | Water + concentrate cells solution 10 fold dilution | ||
Water + concentrate cells solution 100 fold dilution | Water + concentrate cells solution 100 fold dilution | ||
− | |||
J0: we take 100µL of each water and put it on plates with specific antibiotic: | J0: we take 100µL of each water and put it on plates with specific antibiotic: | ||
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We choose to only use MCK+Sorbitol now | We choose to only use MCK+Sorbitol now | ||
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===Low Budget Challenge=== | ===Low Budget Challenge=== | ||
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We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria. | We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria. | ||
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'''Member present:''' | '''Member present:''' |
Latest revision as of 21:30, 18 September 2015
Contents
Tuesday 4th August
Lab Work
Plasmid extraction
by Pauline
- BBa_K1707008 #1 to #6
- BBa_I13600 #1 and #2
With the Macherey-Nagel Extraction kit
Digestion Verification
by Pauline and Coralie
- BBa_K1707008 #1 to #6
Mix:
- 2 µL Plasmid
- 0,5 µL XbaI
- 0,5 µL PstI
- 1 µL Buffer FastDigest 10x
- 6 µL H2O
Incubation 2h, 37°C
Electrophoresis
by Pauline
Agarose gel 1% Migration 90V
Verification by digestion with XbaI and PstI of BBa_K1707008, from left to right: 1. DNA Ladder, 2. #1, 3. #2, 4. #3, 5. #4, 6. #5, 7. #6, 8. Empty, 9. Empty, 10. Empty, 11. Empty, 12. Empty
We can conclude that all BBa_K1707008 clones are OKWe make a glycerol stock of #1 and #2
Electrophoresis Verification
by Pauline and Coralie
Agarose gel, 1%
Migration 100V
Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707021#1, 3. BBa_K1707021#2, 4. BBa_K1707021#3, 5. BBa_K1707021#4, 6. BBa_K1707021#5, 7. BBa_K1707021#6, 8. BBa_K1707011#4, 9. BBa_K1707011#5, 10. BBa_K1707011#6
Verification, from left to right: 1. DNA Ladder, 2. BBa_K1707004#1, 3. BBa_K1707004#2, 4. BBa_K1707004#3, 5. BBa_K1707004#4, 6. BBa_K1707004#5, 7. BBa_K1707004#6, 8. BBa_K1707011#1, 9. BBa_K1707011#2, 10. BBa_K1707011#3, 11. Empty, 12. Empty
We can conclude that:- BBa_K1707021: #2, #3, #4 and #6 are OK
- BBa_K1707011: #1 to #6 are OK
- BBa_K1707004: #1 to #6 are OK
We make glycerol stock of them.
Digestion
by Coralie
XbaI + PstI:
- BBa_K1707011
- BBa_K1707004 x2
- BBa_E0022
- BBa_E0422
- BBa_K1707012
SpeI + Pst:
- BBa_R0051 x2
- BBa_K1707013
- BBa_K1707019
- BBa_K1707000
XbaI + EcoRI:
- BBa_I13602
EcoRI + SpeI:
- BBa_K1707004
Mix:
- 1 µL Enzyme 1
- 1 µL Enzyme 2
- 2 µL Buffer FastDigest 10x
- 6 µL H2O
- 10 µL Plasmide
Purification agarose gel
by Coralie and Audrey
- BBa_K1707011
- BBa_K1707004
- BBa_E0022
- BBa_E0422
- BBa_K1707012
- BBa_I13602
Agarose gel 1%
Migration 100 V
Verification of gel purification, from left to right: 1. DNA Ladder, 2. BBa_K1707011, 3. BBa_K1707004, 4. BBa_K1707004, 5. BBa_E0022, 6. BBa_E0422, 7. BBa_K1707012, 8. BBa_I13602, 9. Empty, 10. Empty
We can conclude that I13602 isn't digestedPurification
by Pauline
- BBa_K1707011
- BBa_K1707004
- BBa_E0022
- BBa_E0422
- BBa_K1707012
- BBa_R0051
- BBa_K1707013
- BBa_K1707019
- BBa_K1707000
- BBa_K1707004
With Macherey-Nagel Kit
Soil experiment
by Coralie
Water
Sea Water separate in 5mL in 50mL Falcon Fresh water separate in 5mL in 50mL Falcon Contamination with 1mL Water + LB Water + concentrate cells solution Water + concentrate cells solution 10 fold dilution Water + concentrate cells solution 100 fold dilution
J0: we take 100µL of each water and put it on plates with specific antibiotic:
- MacConkey + Spectinomycin
- MacConkey + Tetracyclin
- MacConkey + Chloramphenicol
And only MacConkey for the Water + LB
Incubation ON, 37°C
Soil
Observation of plates (03/08/2015):
Less contamination on plates with MCK+Sorbitol thant MCK+Lactose.
We choose to only use MCK+Sorbitol now
Low Budget Challenge
by Coralie
Observation of plates (03/08/2015): We can saw a little bit of colony. We can suppose that the initial culture wasn't enough in growing phase.
We try another time, with the same protocol than the 03/08/2015 but a fresh culture of bacteria.
Member present:
- Instructors: Claire
- Students: Coralie, Audrey and Pauline