Team:Paris/September 11

From 2008.igem.org

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==Ligation==
==Ligation==
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 +
3 ligases tested
 +
*ligase 1: our 250 µL (400 000 U/mL) tube
 +
*ligase 2: our 50 µL (400 000 U/mL) tube
 +
*ligase 3: 2nd floor 50 µL (400 000 U/mL) tube
 +
 +
{|border="1" style="text-align: center"
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|'''ligation'''
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|1
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|2
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|3
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|4
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|5
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|6
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|7
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|8
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|9
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|10
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|11
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|12
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|13
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|-
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|'''digested DNA used'''
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|colspan="7"|D202 (EcoRI digestion)
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|colspan="6"|D203 (BspHI digestion)
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|-
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|'''type of purification <br> of the digestion products
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|colspan="3"|gel extraction
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|colspan="3"|column
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|no purification
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|colspan="3"|column
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|colspan="3"|no purification
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|-
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|'''ligase used'''
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|L1
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|L2
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|L3
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|L1
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|L2
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|L3
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|L1
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|L1
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|L2
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|L3
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|L1
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|L2
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|L3
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|}

Revision as of 13:04, 12 September 2008

← Yesterday

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Tomorrow →

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 3 kb 3 kb 1,9 kb
1 kb

Purification

After purification
well n°2: D202 purified by gel
well n°3: D202 purified by column
well n°4: D203 purified by column
well n°5: MP101

D202 (EcoRI digestion) was purified in 2 ways:

  • by gel extraction
  • or by column

D203 (BspHI digestion) was purified:

  • by column

Elution in 30 µL of EB buffer

Ligation

3 ligases tested

  • ligase 1: our 250 µL (400 000 U/mL) tube
  • ligase 2: our 50 µL (400 000 U/mL) tube
  • ligase 3: 2nd floor 50 µL (400 000 U/mL) tube
ligation 1 2 3 4 5 6 7 8 9 10 11 12 13
digested DNA used D202 (EcoRI digestion) D203 (BspHI digestion)
type of purification
of the digestion products 		gel extraction column no purification column no purification
ligase used L1 L2 L3 L1 L2 L3 L1 L1 L2 L3 L1 L2 L3
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