This is who we are.

The Molecular Arrhytmia Research (MolAR) group was started two years ago at the University of Szeged to provide a framework for our efforts to combine modern molecular biology techniques with the cellular and in vivo electrophysiology approaches. We aim to dig deeper into the pathomechanisms of cardiac arrhythmias, with particular attention payed on ion channel disfunctions. Eventhough the impudent young age of our group, we like to think that significant amount of know-how of this specific blend of techniques has already been accumulated, which may be of the interest of many of you. On these pages we would like to share the protocols, recepies and other handy stuff that we use, follow and depend on in our lab. Hope you will enjoy reading and that you will find some cool and usefull stuff.

The Bench Routine section will list the protocols we use, completed by adding tips and tricks.

MolAR proceedings will cover the hopefully fast-paced progress of our research activities.

Under Ideas, we set our minds free under the motto: nothing is impossible.

So, see you soon!

Balazs Ordog

Plasmid minipreps. So basic, so simple, so beautiful!

This is how we purify plasmid DNA!  No kits please!

1)      Inoculate bacterial culture aseptically in 3 ml LB (or similar media) with the appropriate antibiotics.  Incubate over night with agitation (250-300 rpm) @ 37 ^oC.

           Inoculation means picking a colony with an inoculation loop.  Or if you don`t have one, take sterile forceps and pick a sterile pipette tip or tooth stick or anything else that is sterile and have a fine tip.  Tap onto the colony and drop it into the tube with LB.  Replica plates can also be produced at this step, just scratch the surface of a fresh plate with loop/pipette tip/tooth stick and then drop it into the tube.  And once you are already at making replica plates, why not pool the minipreps? When working with difficult ligations, we pool three colonies per tube. Just make sure you replicate each of them on the replica plate with proper labeling!

2)      Transfer 2 ml of the over night culture into 2 ml Eppendorf tube.

Also works with 1.5 ml culture in 1.5 ml tubes.  Transfer means pouring, you don`t have to keep this sterile.

3)      Pellet the cells by centrifugation (~7-8000 g, 1 min).

4)      Remove supernatant and re-suspend cells in 300 μl P1 by vortexing.

5)      Add 300 μl P2.  Turn the tubes upside down and back 8 times.  (Do not vortex or shake too hard!  You can add solution first to each tube, close each tube and then make the mixing).

6)      Let the tubes stand on the bench for 5 mins.

7)      Add 300 μl P3, close the cap, shake the tube and place it on ice. (Shake the tube vigorously immediately after adding solution, but do not vortex!)

8)      Let the tubes stand on ice for 10 mins.  (5 mins is enough when you are on a rush.  You can also extend this incubation for up to an hour.)

9)      Centrifuge at 12000 g for 10 mins.

10)  Transfer supernatant into a new 1.5 ml Eppendorf tube.

11)  Add 200 μl phenol-chlorophorm, mix by vortexing.

12)  Centrifuge at 12000 g for 10 mins.

13)  Transfer supernatant into a new 1.5 ml Eppendorf tube.

14)  Add 500 μl i-propanol.  Mix and let the tube stand on the bench for 5 mins.

15)  Centrifuge at 12000 g for 10 mins.

16)  Remove supernatant and add 500 μl 70% Et-OH.

17)  Centrifuge at 12000 g for 10 mins.

18)  Remove supernatant and dry the pellet.

19)  Add 50 μl ultra pure water with 10 μg / ml RNase.

Solutions

P1

Dissolve 6.06 g Tris base, 3.72 g Na₂EDTA.2H₂O in 800 ml distilled water. Adjust the pH to 8.0 with HCl. Adjust the volume to 1 liter with distilled water.

Optionally 100 mg RNase A per liter can be added. Store at 4^oC.

P2

Dissolve 8 g NaOH and 10 g SDS (1 m/w %) in 1 liter final volume of water. Store at room temperature.

P3

Dissolve 294.5 g potassium acetate in 500 ml distilled water. Adjust pH to 5.5 with glacial acetic acid (110-130 ml). Adjust volume to 1 liter with distilled water. Store at 4^oC.