Team:Paris/September 12

From 2008.igem.org

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(→Checking our ligases (following))
(→Checking our ligases (following))
 
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==Analysis of the ligation products by electrophosesis==
==Analysis of the ligation products by electrophosesis==
-
 
-
[[Image:Ligation-D202.JPG|thumb|Ligation of the plasmid digested by EcoRI]]
 
-
[[Image:Ligation-D203.JPG|thumb|Ligation of the plasmid digested by BspHI]]
 
1% agarose gel
1% agarose gel
 +
 +
[[Image:Ligation-D202.JPG|thumb|Ligation of the plasmid digested by '''EcoRI''']]
 +
[[Image:Ligation-D203.JPG|thumb|Ligation of the plasmid digested by '''BspHI''']]
'''Analysis of the ligation of the plasmid digested by EcoRI'''
'''Analysis of the ligation of the plasmid digested by EcoRI'''
 +
<br>No heat inactivation of the ligase
{|border="1" style="text-align: center"
{|border="1" style="text-align: center"
|'''well n°'''
|'''well n°'''
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|-
|-
|'''sample'''
|'''sample'''
-
|1 kb ladder
+
|rowspan="3"|1 kb ladder
-
|MP101 <br> 0,5 µL
+
|rowspan="3"|circular <br> plasmid <br> (MP101)
-
|MP101 <br> 1 µL
+
|rowspan="3"|circular <br> plasmid <br> (MP101)
-
|D202 <br> 2 µL
+
|rowspan="3"|digested <br> plasmid <br> (D202)
-
|D202 <br> 3 µL
+
|rowspan="3"|digested <br> plasmid <br> (D202)
 +
|'''1'''
 +
|'''2'''
 +
|'''3'''
 +
|'''4'''
 +
|'''5'''
 +
|'''6'''
 +
|'''7'''
 +
|rowspan="3"|100 bp ladder
 +
|-
 +
|'''purification of <br> digestion products'''
 +
|colspan="3"|gel extraction
 +
|colspan="3"|column
 +
|no <br> purification
 +
|-
 +
|'''ligase used'''
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|-
 +
|'''amount loaded'''
 +
|10 µL
 +
|0,5 µL
 +
|1 µL
 +
|2 µL
 +
|3 µL
 +
|colspan="6"|10 µL
 +
|3 µL
 +
|10 µL
 +
|}
 +
 
 +
<br>
 +
'''Analysis of the ligation of the plasmid digested by BspHI'''
 +
<br>Heat inactivation of the ligase (20 min at 65°C)
 +
{|border="1" style="text-align: center"
 +
|'''well n°'''
|1
|1
|2
|2
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|6
|6
|7
|7
-
|100 bp ladder
+
|8
 +
|9
 +
|10
 +
|11
 +
|-
 +
|'''sample'''
 +
|rowspan="3"|1 kb ladder
 +
|rowspan="3"|circular <br> plasmid <br> (MP101)
 +
|rowspan="3"|circular <br> plasmid <br> (MP101)
 +
|rowspan="3"|digested <br> plasmid <br> (D203)
 +
|'''8'''
 +
|'''9'''
 +
|'''10'''
 +
|'''11'''
 +
|'''12'''
 +
|'''13'''
 +
|rowspan="3"|100 bp ladder
 +
|-
 +
|'''purification of <br> digestion products'''
 +
|colspan="3"|column
 +
|colspan="3"|no <br> purification
 +
|-
 +
|'''ligase used'''
 +
|L1
 +
|L2
 +
|L3
 +
|L1
 +
|L2
 +
|L3
 +
|-
 +
|'''amount loaded'''
 +
|10 µL
 +
|0,3 µL
 +
|0,5 µL
 +
|4 µL
 +
|colspan="3"|15 µL
 +
|colspan="3"|10 µL
 +
|10 µL
|}
|}

Latest revision as of 14:16, 12 September 2008

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Checking our ligases (following)

Analysis of the ligation products by electrophosesis

1% agarose gel

Ligation of the plasmid digested by EcoRI
Ligation of the plasmid digested by BspHI

Analysis of the ligation of the plasmid digested by EcoRI
No heat inactivation of the ligase

well n° 1 2 3 4 5 6 7 8 9 10 11 12 13
sample 1 kb ladder circular
plasmid
(MP101) 		circular
plasmid
(MP101) 		digested
plasmid
(D202) 		digested
plasmid
(D202) 		1 2 3 4 5 6 7 100 bp ladder
purification of
digestion products 		gel extraction column no
purification
ligase used L1 L2 L3 L1 L2 L3 L1
amount loaded 10 µL 0,5 µL 1 µL 2 µL 3 µL 10 µL 3 µL 10 µL


Analysis of the ligation of the plasmid digested by BspHI
Heat inactivation of the ligase (20 min at 65°C)

well n° 1 2 3 4 5 6 7 8 9 10 11
sample 1 kb ladder circular
plasmid
(MP101) 		circular
plasmid
(MP101) 		digested
plasmid
(D203) 		8 9 10 11 12 13 100 bp ladder
purification of
digestion products 		column no
purification
ligase used L1 L2 L3 L1 L2 L3
amount loaded 10 µL 0,3 µL 0,5 µL 4 µL 15 µL 10 µL 10 µL
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