Revision as of 18:03, 9 September 2015 (view source) Coralie (Talk | contribs) ← Older edit		Revision as of 18:12, 9 September 2015 (view source) Coralie (Talk | contribs) Newer edit →
Line 135:		Line 135:
	''by Coralie''		''by Coralie''
−	We make 8 plates of home	+	We make 8 plates:
		+	* In each one:
		+	** 0,27g Stock Cube
		+	** 0,600g Agar Agar
		+	** 0,135g Baker's yeast
		+	** 45mL mineral water
		+	We make 3 LB plates for control
		+	We use the strain created for the Interlab Study (BBa_J23101 + GFP) to make stries on each plate.
		+	We put 3 plates in each condition (2 home-made + 1 LB plate)
		+	* 37°C
		+	* In a yogurt maker
		+	* On the table
−		+	Incubation overnight
−		+
−		+
−		+
−	===Digestion verification===	+
−	''by Coralie''	+
−		+
−	Biobricks:	+
−	* BBa_K1707004 #2	+
−	* BBa_K1707011 #1	+
−	* BBa_ROO407#1	+
−	* BBa_K1707006 #1	+
−	* BBa_I13602 #1	+
−	* BBa_K1707012 #1	+
−	* BBa_R0051 #1	+
−	* BBa_S03518 #1	+
−		+
−		+
−	* Mix for BBa_S03518 and BBa_K1707006:	+
−	** 10 µL plasmid	+
−	** 1 µL XbaI	+
−	** 1 µL PstI	+
−	** 2 µL Buffer FastDigest 10x	+
−	** 6 µL H2O	+
−		+
−		+
−	* Mix for BBa_K1707011 and BBa_K1707012:	+
−	** 15 µL plasmid	+
−	** 1 µL XbaI	+
−	** 1 µL PstI	+
−	** 2 µL Buffer FastDigest 10x	+
−	** 1 µL H2O	+
−		+
−		+
−	* Mix for BBa_R0040 and BBa_I13602:	+
−	** 10 µL plasmid	+
−	** 1 µL EcoRI	+
−	** 1 µL XbaI	+
−	** 2 µL Buffer FastDigest 10x	+
−	** 6 µL H2O	+
−		+
−		+
−	* Mix for BBa_R0051	+
−	** 10 µL plasmid	+
−	** 1 µL SpeI	+
−	** 2 µL PstI	+
−	** 2 µL Tango Buffer 10x	+
−	** 6 µL H2O	+
−		+
−	Incubation 1h30, 37°C	+
−		+
−		+
−	* Mix for BBa_K1707004:	+
−	** 2 µL Tango Buffer 10x	+
−	** 1 µL  SpeI	+
−	** 2 µL H2O	+
−	** 15 µL  plasmid	+
−		+
−	Incubation 1h30, 37°C	+
−	After incubation, we add:	+
−	** 3 µL Tango Buffer 10x	+
−	** 1 µL EcoRI	+
−	** 1 µL H2O	+
−	Incubation 1h30, 37°C	+
−		+
−		+
−	===Purification by Electrophoresis===	+
−	''by Coralie''	+
−		+
−	Agarose gel 1%, 100V	+
−		+
−	Biobricks:	+
−	* BBa_K1707004 #1 and #2 (digested by EcoRI + SpeI)	+
−	* BBa_K1707011 #1 (digested by XbaI +PstI)	+
−	* BBa_K1707006 #1 (digested by XbaI + PstI)	+
−	* BBa_K1707012 #1 (digested by XbaI + PstI)	+
−	* BBa_S03518 #1 (digested by XbaI + PstI)	+
−		+
−		+
−	We can conclude that:	+
−	* BBa_K1707004 #1 and #2  are OK, we can cut the band	+
−	* BBa_K1707006  is OK, we can cut the band	+
−	* BBa_K1707011: we can't see anything on the gel	+
−	* BBa_K1707012: we can't see anything on the gel	+
−	* BBa_S03518: is OK, we can cut the band	+
−		+
−		+
−	===Purification===	+
−	''by Pauline''	+
−		+
−	Biobricks:	+
−	* BBa_K1707004 #2	+
−	* BBa_K1707011 #1	+
−	* BBa_K1707006 #1	+
−	* BBa_K1707012 #1	+
−	* BBa_S03518 #1	+
−	* BBa_R0040 #1	+
−	* BBa_I13602 #1	+
−	* BBa_R0051 #1	+
−		+
−	With Macherey-Nagel Purification kit	+
−		+
−		+
−	===Quantification===	+
−	''by Pauline''	+
−		+
−	Biobricks:	+
−	* BBa_K1707004 #2 x2	+
−	* BBa_K1707006 #1	+
−	* BBa_S03518 #1	+
−	* BBa_R0040 #1	+
−	* BBa_I13602 #1	+
−	* BBa_R0051 #1	+
−		+
−	Agarose gel 1%, migration at 100V	+
−		+
−	We can conclude:	+
−	* BBa_K1707004 #2: 20µg/µL	+
−	* BBa_K1707006 #1: 30µg/µL	+
−	* BBa_S03518 #1: 50µg/µL	+
−	* BBa_R0040 #1: 30 µg/µL	+
−	* BBa_I13602 #1: 50µg/µL	+
−	* BBa_R0051 #1: we can't see anything	+
−		+
−		+
−	===New Culture===	+
−	''by Pauline''	+
−		+
−	Biobricks:	+
−	* BBa_K1707026	+
−	* BBa_K1707032	+
−		+
−	We put two clones of each in 5mL LB + 5µL Chloramphenicol	+

---

Revision as of 18:12, 9 September 2015

Tuesday 11th August

Lab Work

Ligation

by Pauline

• BBa_K1707031: BBa_K1707004 + BBa_R0040
  • 10,4 µL BBa_K1707004 digested by EcoRI + SpeI
  • 3,3 µL BBa_R0040 digested by EcoRI + XbaI
  • 2 µL Ligase
  • 2 µL Buffer 10x
  • 2,3 µL H2O

• BBa_K1707033: BBa_K1707006 + BBa_B0030
  • 7,3 µL BBa_K1707006 digested by XbaII + PstI
  • 7,4 µL BBa_B0030
  • 2 µL Ligase
  • 2,5 µL Buffer 10x
  • 5,8 µL H2O

• BBa_K1707016: BBa_K1707004 + BBa_I13602
  • 7,2 µL BBa_K1707004 digested by EcoRI + SpeI
  • 2 µL BBa_B0030
  • 2 µL Ligase
  • 1,5 µL Buffer 10x
  • 2,5 µL H2O

Incubation 4°C, 3h

Plamid Extraction

by Pauline

Biobricks:

• BBa_K1707026 #1 and #2
• BBa_K1707032 #1 and #2

With Macherey-Nagel Extraction kit

Digestion

by Coralie

Biobricks:

• BBa_K1707026 #1 and #2
• BBa_K1707032 #1 and #2

Mix for each reaction:

• 0,5 µL PstI
• 0,5 µL XbaI
• 1 µL Buffer FastDigest 10x
• 6 µL H2O
• 2 µL plasmid

Incubation 37°C, 2h

Electrophoresis

by Pauline

Agarose gel 1%, migration 90V

Biobricks:

• Quantification:
  • BBa_K1707011 #3 (plasmid)
  • BBa_K1707012 #2 (plasmid)
• Verification of digestion:
  • BBa_K1707026 #1
  • BBa_K1707026 #2
  • BBa_K1707028 #1
  • BBa_K1707028 #2
  • BBa_K1707032 #1
  • BBa_K1707032 #2

We can conclude:

• Quantification:
  • BBa_K1707011 #3 (plasmid): 13 µg/µL
  • BBa_K1707012 #2 (plasmid): 20 µg/µL
• Verification of digestion:
  • BBa_K1707026 #1: OK but not totally digested
  • BBa_K1707026 #2: OK but not totally digested
  • BBa_K1707028 #1: OK
  • BBa_K1707028 #2: OK
  • BBa_K1707032 #1: OK but not totally digested
  • BBa_K1707032 #2: OK but not totally digested

Digestion

by Coralie

Biobricks:

• BBa_I13602 x2
  • Mix for each reaction:
    • 1 µL EcoRI
    • 1 µL XbaI
    • 2 µL Buffer FastDigest 10x
    • 6 µL H2O
    • 10 µL plasmid
  • Incubation 37°C, 2h

• BBa_K1707030 and BBa_K1707020
  • Mix for each reaction:
    • 2 µL Tango Buffer
    • 1 µL SpeI
    • 7 µL H2O
    • 10 µL Plasmid
  • Incubation 2h, 37°C
  • After incubation: we add in each tube:
    • 3 µL Tango Buffer
    • 1 µL EcoRI
    • 1 µL H2O
  • Incubation 1h30, 37°C

Purification on gel

by Pauline

Biobricks:

• BBa_K1707020
• BBa_K1707030

We can conclude that biobricks are OK

Transformation

by Pauline

Biobricks:

• BBa_K1707016
• BBa_K1707031
• BBa_K1707033

As usual

Low cost experiment

by Coralie

We make 8 plates:

• In each one:
  • 0,27g Stock Cube
  • 0,600g Agar Agar
  • 0,135g Baker's yeast
  • 45mL mineral water

We make 3 LB plates for control

We use the strain created for the Interlab Study (BBa_J23101 + GFP) to make stries on each plate. We put 3 plates in each condition (2 home-made + 1 LB plate)

• 37°C
• In a yogurt maker
• On the table

Incubation overnight

Member present:

• Instructors: Claire
• Students: Coralie and Pauline

Back to the calendar