Revision as of 15:10, 7 August 2015 (view source) JohanB (Talk | contribs) (Created page with "=Wednesday 5th August= ==Lab Work== ===Plasmid extraction=== ''by Pauline'' Biobricks: * BBa_K1707008 #1 to #6 * BBa_I13600 #1 and #2 With the Macherey-Nagel Extraction kit ==...")		Revision as of 15:28, 7 August 2015 (view source) JohanB (Talk | contribs) (→‎Wednesday 5th August) Newer edit →
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	=Wednesday 5th August=		=Wednesday 5th August=
	==Lab Work==		==Lab Work==
−	===Plasmid extraction===	+	===Quantification===
−	''by Pauline''	+	''by Pauline and Coralie''
	Biobricks:		Biobricks:
−	* BBa_K1707008 #1 to #6	+	* BBa_R0051
−	* BBa_I13600 #1 and #2	+	* BBa_E0022 (15ng/mL)
		+	* BBa_E0422 (20ng/mL)
		+	* BBa_K1707000 (20ng/mL)
		+	* BBa_K1707004 (15ng/mL)
		+	* BBa_K1707011
		+	* BBa_K1707012
		+	* BBa_K1707013 (20ng/mL)
		+	* BBa_K1707019 (15ng/mL)
−	With the Macherey-Nagel Extraction kit	+	Agarose gel, 1%
		+	Migration 90V
−	===Digestion Verification===
−	''by Pauline and Coralie''
−	Biobricks: BBa_K1707008 #1 to #6	+	===Ligation===
		+	''by Pauline''
		+
		+	Biobricks:
		+	* BBa_K1707020 : BBa_K1707004 + BBa_K1707019
	Mix:		Mix:
−	* 2 µL Plasmid	+	* 6,5 µL BBa_K1707019
−	* 0,5 µL XbaI	+	* 9,5 µL BBa_K1707004
−	* 0,5 µL PstI	+	* 2 µL Tampon 10x
−	* 1 µL Buffer FastDigest 10x	+	* 1 µL Ligase
−	* 6 µL H2O	+	* 1 µL H2O
−	Incubation 2h, 37°C	+	* BBa Test : BBa_K1707004 + BBa_K1707013
		+	Mix:
		+	* 5 µL BBa_K1707013
		+	* 7,5 µL BBa_K1707004
		+	* 1,5 µL Tampon 10x
		+	* 1 µL Ligase
−	===Electrophoresis===	+	* BBa_K1707027 : BBa_K1707000 + BBa_E0022
−	''by Pauline''	+
−	Agarose gel 1%	+	Mix:
−	Migration 90V	+	* 5 µL BBa_K1707000
		+	* 9 µL BBa_E0022
		+	* 1,5 µL Tampon 10x
		+	* 1 µL Ligase
		+	* 3,5 µL H2O
−	We can conclude that all BBa_K1707008 clones are OK	+	* BBa_K1707028 : BBa_R0051 + BBa_E0422
−	We make a glycerol stock of #1 and #2	+
		+	Mix:
		+	* 6,25 µL BBa_R0051
		+	* 5,5 µL BBa_E0422
		+	* 1,5 µL Tampon 10x
		+	* 1 µL Ligase
		+	* 0,75 µL H2O
−	===Electrophoresis===
−	''by Pauline and Coralie''
−	Agarose gel, 1%	+	===Rehydratation===
		+	''by Audrey''
−	Migration 100V	+	BBa_R0040
−	We can conclude that:	+	===Transformation===
−	* BBa_K1707021: #2, #3, #4 and #6 are OK	+	''by Audrey and Coralie''
−	* BBa_K1707011: #1 to #6 are OK	+
−	* BBa_K1707004: #1 to #6 are OK	+
−	We make glycerol stock of them.	+	* BBa_R0040
		+	* BBa Test
		+	* BBa_K1707028
		+	* BBa_K1707027
		+	* BBa_K1707020
	===Digestion===		===Digestion===
−	''by Coralie''	+	''by Pauline''
	XbaI + PstI:		XbaI + PstI:
		+	* BBa_K1707012
	* BBa_K1707011		* BBa_K1707011
−	* BBa_K1707004 x2	+	* BBa_I13600
−	* BBa_E0022	+
−	* BBa_E0422	+
−	* BBa_K1707012	+
−	SpeI + Pst	+	SpeI + Pst:
−	=== New culture===	+	* BBa_R0051
−	'' by Pauline''	+
−		+
−	Biobricks:	+
	* BBa_K1707008		* BBa_K1707008
−	* BBa_I13600 (2014)
−	6 clones of each in 5mL LB + 20µg/mL of chloramphenicol	+	EcoRI + SpeI:
		+	* BBa_K1707004
−	Incubation at 37°C, 250 rpm	+	XbaI + EcoRI:
		+	* BBa_I13602
	===Soil experiment===		===Soil experiment===
Line 91:		Line 111:
	Incubation ON, 37°C		Incubation ON, 37°C
−
−	===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
−
−
−
−
	'''Member present:'''		'''Member present:'''

---

Revision as of 15:28, 7 August 2015

Wednesday 5th August

Lab Work

Quantification

by Pauline and Coralie

Biobricks:

• BBa_R0051
• BBa_E0022 (15ng/mL)
• BBa_E0422 (20ng/mL)
• BBa_K1707000 (20ng/mL)
• BBa_K1707004 (15ng/mL)
• BBa_K1707011
• BBa_K1707012
• BBa_K1707013 (20ng/mL)
• BBa_K1707019 (15ng/mL)

Agarose gel, 1% Migration 90V

Ligation

by Pauline

Biobricks:

• BBa_K1707020 : BBa_K1707004 + BBa_K1707019

Mix:

• 6,5 µL BBa_K1707019
• 9,5 µL BBa_K1707004
• 2 µL Tampon 10x
• 1 µL Ligase
• 1 µL H2O

• BBa Test : BBa_K1707004 + BBa_K1707013

Mix:

• 5 µL BBa_K1707013
• 7,5 µL BBa_K1707004
• 1,5 µL Tampon 10x
• 1 µL Ligase

• BBa_K1707027 : BBa_K1707000 + BBa_E0022

Mix:

• 5 µL BBa_K1707000
• 9 µL BBa_E0022
• 1,5 µL Tampon 10x
• 1 µL Ligase
• 3,5 µL H2O

• BBa_K1707028 : BBa_R0051 + BBa_E0422

Mix:

• 6,25 µL BBa_R0051
• 5,5 µL BBa_E0422
• 1,5 µL Tampon 10x
• 1 µL Ligase
• 0,75 µL H2O

Rehydratation

by Audrey

BBa_R0040

Transformation

by Audrey and Coralie

• BBa_R0040
• BBa Test
• BBa_K1707028
• BBa_K1707027
• BBa_K1707020

Digestion

by Pauline

XbaI + PstI:

• BBa_K1707012
• BBa_K1707011
• BBa_I13600

SpeI + Pst:

• BBa_R0051
• BBa_K1707008

EcoRI + SpeI:

• BBa_K1707004

XbaI + EcoRI:

• BBa_I13602

Soil experiment

by Coralie

Soil

Observation of plates (31/07/2015):

• Soil + H2O on new MCK: lots of colonies are visible
• Soil + LB on new MCK: lots of colonies are visible
• LB on new MCK: nothing
• MCK alone: nothing

We can suppose two different hypothesis:

• 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

Member present:

• Instructors: Claire
• Students: Coralie, Audrey and Pauline

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