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From 2012.igem.org

Weeks:

June			July				August				September				October				November

Week 2: 18th June - 24th June

Monday, 18th June

• Inoculation
• Auxin and salkowski's test purchased

Tuesday, 19th June

Miniprep

• J23100 RFP: 101.9 ng/uL
  
• K515100 Auxin: 319.7 ng/uL
  

Misc

Liquid culture of aux and RFP

Wednesday, 20th June

Previous work :

Liquid culture of DH5a with mRFP and auxin plasmid

Main goals:

1. Auxin toxicity test
2. Bacterial integration assessment into the xenopus embryos

Experiments

1. Xenopus embryos present a protective membrane, we decided to realize our bacterial deposits experiments on xenopus with and without their membrane. Then, we could see if bacterias could enter into the embryos naturally.
2. On 96 plates, each plate contains 1 embryo:
   • Plate 1:
     • Line A & B = on embryos without protective membrane : 500 uL mRFP plasmid on DH5a deposite + 1mL LB + 1mL MMR medium
       
     • Line C : 1 2 3 : normal embryos : 500 uL mRFP plasmid on DH5a deposite + 1mL (LB + MMR medium)
       
     • Line E & F = embryos without protective membrane : 500 uL AUX plasmid integrated on DH5a deposit + 1mL LB + 1mL MMR medium
       
     • Line G & M1-6 = normal embryos : 500 uL AUX plasmid integrated on DH5a deposit + 1mL LB + 1mL MMR medium
       
     • Line M7-9 = normal embryos : controls
   • Plate 2 (normal embryos only):
     • Line A & B
     • Line C : LB/MMR medium
     • Line D : RFP 0.1uL
     • Line E : AUX 0.1uL
     • Line G & H : LB/MMR medium only (no embryos)
     
3. On 16 Plates each plate contains 3 tadpoles:
   • Line A : 0.5uL RFP bacteria
   • Line B1 : 0.3uL RFP bacteria
   
   

We put this plate on UV light 15min after the deposit:

Thursday, 21st June

Results from Wednesday's experiments:

+ = Alive; - = Dead

• Plate 1: All the embryos are dead except H7
• Plate 2:

  L\C	1	2	3	4	5	6	7	8	9	10	11	12
  A	-
  B	-
  C	-
  D	-	-	+	-	+	-	+	+	+	-	+	+
  E	+	-	-	+	-	+	-	-	-	-	+	+

• Plate 3:

  L\C	1	2	3	4
  A	-	+	+	+
  B	-	+	+	+

Comments:

• Plate 2: 50% of dead when we spread bacterias on embryos. All dead if we drown the embryos of bacteria.
• Plate 1: All dead except 1
• Plate 3: All the tadpoles are fluorescent except the control one.

Friday, 22nd June

Bacterial Transformation

Bacteria: T10
DNA: pCS2 (+) at 640 ng.uL-1
Protocol:

1. Keep constantly the cells on ice
2. Add 1 uL of pCS2 (+) in 100 uL of T10
3. Incubate 30 min on ice
4. Heat shock the cells during 30 sec at 42 degree Celsius in a water-bath without shaking
5. Put 2 min on ice
6. Add 500 uL of pre warmed SOC-medium
7. Incubate 1h at 37 degree Celsius at 225 rpm
8. Spin at 5000 rpm during 30 sec
9. Remove 150 uL - 400 uL of supernatant
10. Resuspend the pellet in the 150 uL left
11. Spread on appropriate plates
12. Incubate overnight at 37 degree Celsius

Promoters received:

Spect

• p2 HB9
• p2 Rosa26
• p2 vimentin

Kana

• p2 Flk-1
• p2 Xlurp
• p2 foxi short
• p2 foxi long
• p2 pax3
• p2 Ef1a
• p2 LMO2 short
• p2 LMO2 long
• p2 vast
• p2 NBT
• p2 HSP70
• p2 pax6
• p2 CMV
• p2 brachyury
• p2 14 xUAS E1b-ATG