Team:Sevilla/Week10

IMPROVED PROTOCOLS

Ligation

We are going to assemble small DNA fragments (inserts) with plasmids, so we will use bigger amount of inserts than os plasmid. Our ligation mix contains:

- 1 μL of 10X ligase buffer. It´s important to keep it on ice and also to resuspend it very well before using it. (Ligase buffer must contains ATP).

- 1 μL of ligase(It must be the last thing you add to the mix). You must not sink the tip too deep in order not to carry much glycerol. KEEP IT IN THE FREEZER, even when you use it.

- 1 or 2 μL of plasmid.

- 7 or 6 μL of insert.

Keep at room temperature overnight.

Double Digestion

20 μL must be the final volumen of our digestion mix. We must add:

- 2 μL of buffer

- 0,5 μL of each enzyme (We use EcorI, SpeI, XbaI and PstI). This amount of enzyme must be individually determine depending on the amunt of DNA you are going to digest

- 16 μL of Milli-Q water

- 1 μL of DNA sample

It´s important that you shake the mixture using the centriguge. 1 minute at medium speed must works.

Transformtion

1. In the same tube, mix:

- 25 μL of competent cells.

- 1 μL of DNA sample (the amount of DNA depends on the concentration of your sample)if yor DNA is pure. If not, use aproximately 3 μL.

*Use very cold pipets.

2. Incubate the tube on ice for 30 minutes.

3. Incubate at 42ºC for 30 seconds.

4. Incubete 2 minutes on ice for 2 minutes.p>

5. Add 225 μL of NZY, LB, or SOB (preheated in the 42 degrees bath). You must add 225 μL of medium culture per each microlitre of DNA sample you use.

6. Keep the tube for 45 minutes or 1h in agitacion at 37ºC

**If you are using complex DNA ligations for the transformation, you must add beta-mercaptoethanol before the half an hour incubation on ice