Revision as of 15:56, 7 August 2015 (view source) JohanB (Talk | contribs) (→‎Water contamination) ← Older edit		Revision as of 09:33, 10 August 2015 (view source) Coralie (Talk | contribs) Newer edit →
Line 32:		Line 32:
	We can conclude that all BBa_K1707008 clones are OK		We can conclude that all BBa_K1707008 clones are OK
		+
	We make a glycerol stock of #1 and #2		We make a glycerol stock of #1 and #2
−	===Electrophoresis===	+	===Electrophoresis Verification===
	''by Pauline and Coralie''		''by Pauline and Coralie''
Line 59:		Line 60:
	* BBa_K1707012		* BBa_K1707012
−	SpeI + Pst	+	SpeI + Pst:
−	=== New culture===	+	* BBa_R0051 x2
−	'' by Pauline''	+	* 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''
	Biobricks:		Biobricks:
−	* BBa_K1707008	+	* BBa_K1707011
−	* BBa_I13600 (2014)	+	* BBa_K1707004
		+	* BBa_E0022
		+	* BBa_E0422
		+	* BBa_K1707012
		+	* BBa_I13602
−	6 clones of each in 5mL LB + 20µg/mL of chloramphenicol	+	Agarose gel 1%
		+
		+	Migration 100 V
		+
		+	We can conclude that I13602 isn't digested
		+
		+
		+	===Purification===
		+	''by Pauline''
		+
		+	Biobricks:
		+	* BBa_K1707011
		+	* BBa_K1707004
		+	* BBa_E0022
		+	* BBa_E0422
		+	* BBa_K1707012
		+	* BBa_R0051
		+	* BBa_K1707013
		+	* BBa_K1707019
		+	* BBa_K1707000
		+	* BBa_K1707004
−	Incubation at 37°C, 250 rpm
	===Soil experiment===		===Soil experiment===
Line 81:		Line 125:
	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
−	We use specific antibiotic for each strain.	+
		+
		+	J0: we take 100µL of each water and put it on plates with specific antibiotic:
	* MacConkey + Spectinomycin		* MacConkey + Spectinomycin
	* MacConkey + Tetracyclin		* MacConkey + Tetracyclin
	* MacConkey + Chloramphenicol		* MacConkey + Chloramphenicol
−	And Just MacConkey for the Water + LB	+
		+	And only MacConkey for the Water + LB
		+
		+	Incubation ON, 37°C
	====Soil====		====Soil====
−	Observation of plates (31/07/2015):	+	Observation of plates (03/08/2015):
−	* Soil + H2O on new MCK:  lots of colonies are visible	+	Less contamination on plates with MCK+Sorbitol thant MCK+Lactose.
−	* Soil + LB on new MCK: lots of colonies are visible	+
−	* LB on new MCK: nothing	+
−	* MCK alone: nothing	+
−	We can suppose two different hypothesis:	+	We choose to only use MCK+Sorbitol now
−	* The soil is contaminated	+
−	* the MCK isn't OK for our experiment: now we add lactose in the preparation. We will try with sorbitol instead of lactose.	+
−	We try soil + H2O and soil +LB on:
−	* MCK + Sorbitol: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol
−	* MCK + Lactose: Without antibiotics, with Spectinomycine, Tetramycine or Chloramphenicol
−	Incubation ON, 37°C	+	===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.
−	===Digestion===
−	''by Audrey and Coralie''
−	Biobricks:
−	* BBa_K1707011 #1 to #6
−	* BBa_K1707021 #1 to #6
−	* BBa_K1707004 #1 to #6
−	Mix:
−	* 2 µL Plasmid
−	* 1 µL Buffer FastDigest 10x
−	* 0,5 µL XbaI
−	* 0,5 µL PstI
−	* 6 µL H2O

---

Revision as of 09:33, 10 August 2015

Tuesday 4th August

Lab Work

Plasmid extraction

by Pauline

Biobricks:

• BBa_K1707008 #1 to #6
• BBa_I13600 #1 and #2

With the Macherey-Nagel Extraction kit

Digestion Verification

by Pauline and Coralie

Biobricks: 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

We can conclude that all BBa_K1707008 clones are OK

We make a glycerol stock of #1 and #2

Electrophoresis Verification

by Pauline and Coralie

Agarose gel, 1%

Migration 100V

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

Biobricks:

• BBa_K1707011
• BBa_K1707004
• BBa_E0022
• BBa_E0422
• BBa_K1707012
• BBa_I13602

Agarose gel 1%

Migration 100 V

We can conclude that I13602 isn't digested

Purification

by Pauline

Biobricks:

• BBa_K1707011
• BBa_K1707004
• BBa_E0022
• BBa_E0422
• BBa_K1707012
• BBa_R0051
• BBa_K1707013
• BBa_K1707019
• BBa_K1707000
• BBa_K1707004

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

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