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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
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
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| D202
10 µL
| D202
10 µL
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| D203
3 µL
| 100 bp ladder
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| expected size
|
|
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| 2984 bp
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| 1870 bp
1008 bp
105 bp
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|
| measured size
|
|
|
| 3 kb
| 3 kb
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| 1,9 kb
1 kb
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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:
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
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| digested DNA used
| D202 (EcoRI digestion)
| D203 (BspHI digestion)
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type of purification
of the digestion products
| gel extraction
| column
| no purification
| column
| no purification
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| ligase used
| L1
| L2
| L3
| L1
| L2
| L3
| L1
| L1
| L2
| L3
| L1
| L2
| L3
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