Team:Paris/September 11

=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 2h30 at 37°C and then 20 min at 65°C
 * 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

Purification


D202 (EcoRI digestion) was purified in 2 ways: D203 (BspHI digestion) was purified: Elution in 30 µL of EB buffer
 * by gel extraction
 * or by column
 * by column

Ligation
3 ligases tested 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
 * 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
 * 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.