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

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.