Team:Paris/September 11

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=Checking our ligases=
=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==
==Digestion==
-
[[Image:Before-excision.JPG|Before excision]]
+
[[Image:Before-excision.JPG|thumb|'''Before excision''']]
-
[[Image:After-excision.JPG|After ecxision]]
+
 
 +
DNA used for digestion: MP101
 +
<br> 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
 +
 
 +
{|border="1" style="text-align: center"
 +
|'''well n°'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|-
 +
|'''sample'''
 +
|1 kb ladder
 +
|MP101 <br> 1 µL
 +
| 
 +
|D202 <br> 10 µL
 +
|D202 <br> 10 µL
 +
| 
 +
|D203 <br> 3 µL
 +
|100 bp ladder
 +
|-
 +
|'''expected size'''
 +
|
 +
|
 +
|
 +
|colspan="2"|2984 bp
 +
|
 +
|1870 bp <br> 1008 bp <br> 105 bp
 +
|
 +
|-
 +
|'''measured size'''
 +
|
 +
|
 +
|
 +
|'''3 kb'''
 +
|'''3 kb'''
 +
|
 +
|'''1,9 kb''' <br> '''1 kb'''
 +
|
 +
|}
==Purification==
==Purification==
 +
 +
[[Image:After-purification.JPG|thumb|'''After purification''' <br> well n°2: D202 purified by gel <br> well n°3: D202 purified by column <br> well n°4: D203 purified by column <br> well n°5: MP101]]
 +
 +
D202 (EcoRI digestion) was purified in 2 ways:
 +
*by gel extraction
 +
*or by column
 +
<br>
 +
D203 (BspHI digestion) was purified:
 +
*by column
 +
<br>
 +
Elution in 30 µL of EB buffer
==Ligation==
==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
 +
The ligase 1 and 2 have been used with our own buffer tube, whereas the ligase 3 has been used with the buffer tube from the 2nd floor lab.
 +
<br>
 +
<br>'''Reaction mixture'''
 +
*8 µL of purified digestion products
 +
*2 µL of T4 DNA ligase 10X buffer
 +
*9 µL of water
 +
*1 µL of T4 DNA ligase
 +
 +
<br>For the samples for which the digestion products have not been purified, 1 µL of ligase and 1 µL of ATP (1 mM final concentration) have been added directly in the digestion products (in the digestion buffer 2).
 +
<br>
 +
<br>'''Reaction mixture for unpurified digestion products'''
 +
*8 µL of unpurified
 +
*1 µL of ATP 10 mM (1mM final concentration)
 +
*1 µL of T4 DNA ligase
 +
 +
<br>All the ligation were carried out '''overnight''' at '''4°C'''.
 +
 +
{|border="1" style="text-align: center"
 +
|'''ligation'''
 +
|1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|9
 +
|10
 +
|11
 +
|12
 +
|13
 +
|-
 +
|'''digested DNA used'''
 +
|colspan="7"|D202 (EcoRI digestion)
 +
|colspan="6"|D203 (BspHI digestion)
 +
|-
 +
|'''type of purification <br> of the digestion products
 +
|colspan="3"|gel extraction
 +
|colspan="3"|column
 +
|no <br> purification
 +
|colspan="3"|column
 +
|colspan="3"|no <br> purification
 +
|-
 +
|'''ligase used'''
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|L2
 +
|L3
 +
|}

Latest revision as of 19:34, 16 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

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

The ligase 1 and 2 have been used with our own buffer tube, whereas the ligase 3 has been used with the buffer tube from the 2nd floor lab.

Reaction mixture

  • 8 µL of purified digestion products
  • 2 µL of T4 DNA ligase 10X buffer
  • 9 µL of water
  • 1 µL of T4 DNA ligase


For the samples for which the digestion products have not been purified, 1 µL of ligase and 1 µL of ATP (1 mM final concentration) have been added directly in the digestion products (in the digestion buffer 2).

Reaction mixture for unpurified digestion products

  • 8 µL of unpurified
  • 1 µL of ATP 10 mM (1mM final concentration)
  • 1 µL of T4 DNA ligase


All the ligation were carried out overnight at 4°C.

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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