(Difference between revisions)

Contents 1 MINIPREP 1.1 Materials 1.2 Procedure 2 Transformation 3 Ligation 4 LB & LB agar 5 Electrophoresis Gel

MINIPREP

Materials

1. Resuspension buffer
2. Lysis buffer
3. Neutralization buffer
4. Isopropanol
5. 70% Etanol
6. dH2O
7. Ice (in ice bucket/container)

Procedure

1. Centrifuge for 5 minutes at 5000 rpm 1,5 ml of bacteria culture.
2. Take the pellet and add 250 ul of resuspension buffer and pipet up and down a few times.(make sure that all is properly mixed)
3. Add 250 ul of Lysis buffer and invert the tubes 5 times to mix. Let rest 5 minutes. The mix must turn transparent.
4. Add 250 ul of cold Neutralization buffer and invert the tubes 5 times to mix. The mix must turn turbid.
5. Centrifuge at 13.000 rpm for 15 minutes.
6. Transfer the supernatant to a new clean eppendorf tube.
7. Add isopropanol (the same volume that is in the tube). Mix many times and put it in ice for 20 minutes.
8. Centrifuge at 13.000 rpm for 20 minutes.
9. Eliminate the supernatant (be carefull that the pellet does not go with it)
10. Add 1 ml of etanol 70% and use a vortex to ensure the pellet mixes with the etanol.
11. Centrifuge at 13.000 rpm for 5 minutes.
12. Eliminate all the supernatant with a pipette (be carefull that the pellet does not go with it)
13. Let the tube open until it is dried. The pellet must turn from white to transparent.
14. Add 20 ul of dH2O and freeze.

Transformation

We have E. Coli DH5α strain competent bacteria, with a transformation efficiency of 10

Important! Competent bacteria should always be on ice.

1. If the plasmid comes from the kit or is a product of ligation, add 2μl of plasmidic DNA into 50μl

of competent bacteria. If it’s a plasmid that comes from a miniprep, use a mass in function of

the efficiency of the bacteria.

1. Incubate for 20 min on ice.

1. Heat Shock: 42°C for exactly 1’30’’, then immediately place on ice.

1. Incubate for 5 min on ice.

1. Recovery: Add 950μl of LB without antibiotic, incubate from 30 min to an hour at 37°C

1. Pellet the bacteria by centrigue at 10000rpm for 5 minutes. Discard the supernatant of LB.

Resuspend the bacteria in the remnant LB.

1. Plaque onto plates with LB and the desired antibiotic.

1. Incubate overnight in a stove at 37°C

Ligation

Important! Before starting the ligation process, make sure that the restriction enzymes are inactive.

1. Add 2μl of digested plasmid backbone (or the equivalent of 25ng).

1. Add equimolar amount of EcoR1-HF Spe1 digested fragment (less than 3μl).

1. Add equimolar amount of Xba1 Pst1 digested fragment (less than 3μl).

1. Add 2μl of T4 DNA ligase buffer (final concentration should be 1x).

1. Add 0.5μl of T4 DNA ligase.

1. Add water to 10μl.

1. Ligate at 16°C for 30 min, then heat kill 80°C for 20min.

1. Transform with 1 – 2 μl of product.

LB & LB agar

1. Liquid LB (1 litre)

1. 10g of bacto peptone (peptone, triptone or bactereologic triptone)

1. 5g of yeast extract

1. 10g of NaCl

1. Fill with dH2O

Autoclave.

TIP: if it is not going to be autoclaved in the next few hours water should not be added (because even

when in the fridge it will get contaminated), in which case everything can be measured and left ready to

just add the water.

1. LB agar (1 litre)

1. 10g of bacto peptone (peptone, triptone or bactereologic triptone)

1. 5g of yeast extract

1. 10g of NaCl

1. 15g of agar

1. Fill with dH2O

IMPORTANT: agar does not dissolve if not heated so if Erlen-Meyers are prepared with LB agar, add LB in

liquid form and then add the agar to the Erlen-Meyer according to the volume.

Autoclave.

TIP: if it is not going to be autoclaved in the next few hours water should not be added (because even

when in the fridge it will get contaminated), in which case everything can be measured and left ready to

just add the water.

Electrophoresis Gel

TAE 1x (diludde from TAE stock solution – 50x)

Agarose

Remember to use gloves during the entire process, being mindful not to have direct contact with

ethidium bromide.

1. Add the necessary agarose into an Erlenmeyer flask and then add TAE, and fill with water. The

final concentration of agarose may depend on the type of gel needed, but usually will be 1%.

1. Heat the flask, while covered, in a microwave until completely dissolved (should take no more

than 3 minutes at maximum potency)

1. Add the necessary volume of Ethidium Bromide (about 10 μl for each 100g of gel)

IMPORTANT: Ethidium Bromide is carcinogenic. It should be handled with gloves, and these,

such as anything that has come in direct contact with Ethidium Bromide must be discarded

separately in an assigned discard bag.

1. Pour the melted gel, with Ethidium Bromide, slowly into the gel tray. Add the combs and leave

until solified. Remove the combs. Rinse the flask with abundant water.

1. Place the gel tray inside the gel box being mindful of the direction in which gel will run. Add TAE

1x until it is completely covered.