Team:Paris/September 11

From 2008.igem.org

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(→Checking our ligases)
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DNA used for digestion: MP101
DNA used for digestion: MP101
<br> 2 digestion were carried out:
<br> 2 digestion were carried out:
-
*one with EcoRI
+
*one with EcoRI (D202), that cuts the plasmid only one time
-
*the other with BspHI
+
*the other with BspHI (D203), that cuts the plasmid three times
 +
 
 +
'''Reaction mixture'''
 +
*10 µL of DNA
 +
*6µL of 10X buffer 2
 +
*0,6 µL of 100X BSA
 +
*2 µL of enzyme (EcoRI or BspHI)
 +
*41,4 µL of water
 +
2h30 at 37°C and then 20 min at 65°C
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{|border="1" style="text-align: center"
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|'''sample'''
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|1 kb ladder
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|MP101 <br> 1 µL
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|
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|D202 <br> 10 µL
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|D202 <br> 10 µL
 +
|
 +
|D203 <br> 3 µL
 +
|100 bp ladder
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|-
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|'''expected size'''
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|colspan="2"|2984 bp
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|
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|1870 bp <br> 1008 bp <br> 105 bp
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|
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|-
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|'''measured size'''
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Revision as of 10:50, 12 September 2008

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Contents

Checking our ligases

Because we couldn't be sure of the results of yesterday experiment, we decided to carry it out one more time.

Digestion

Before excision
After ecxision

DNA used for digestion: MP101
2 digestion were carried out:

  • one with EcoRI (D202), that cuts the plasmid only one time
  • the other with BspHI (D203), that cuts the plasmid three times

Reaction mixture

  • 10 µL of DNA
  • 6µL of 10X buffer 2
  • 0,6 µL of 100X BSA
  • 2 µL of enzyme (EcoRI or BspHI)
  • 41,4 µL of water

2h30 at 37°C and then 20 min at 65°C

well n° 1 2 3 4 5 6 7 8
sample 1 kb ladder MP101
1 µL 		D202
10 µL 		D202
10 µL 		D203
3 µL 		100 bp ladder
expected size 2984 bp 1870 bp
1008 bp
105 bp
measured size

Purification

Ligation
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