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

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

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 product

• 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.