• Wear gloves. Remember,.. they serve to protect your DNA samples.
• ALWAYS use a fresh tip for each step!!
• We have premelted your agarose and they will be waiting for you in a 65oC waterbath. Normally, you will have to melt it yourself in the microwave or using a hot plate. Take care doing this as overboiling can lead to spills. When you do melt the agarose yourself, you must make sure that it has completely dissolved. Look closely to observe for filaments in the solution.

1. You will personally prepare one real digest and one negative control mock digest. The only difference between these two samples is that one will have the restriction enzyme, and the other will not.

-Working in tube F (which contains the 1.0ul of restriction enzyme), add 2.0ul of 10x reaction buffer, and 17ul of lamdba DNA from tube A (Pipette 2.0ul of tube E into tube F. Pipette 17ul of tube A into tube F. Now buffer, enzyme and DNA is all in tube F). This is your digest.
- Add 2.0ul of 10x reaction buffer into another lambda DNA sample (tube B). (Pipette 2.0ul of tube E into tube B). This is the negative control. It is identical to your other sample except that there is NO enzyme.

2. Incubate your two samples (tube F and tube B) in the 37oC for a minimum of 1hr.

3. Add 4ul of the blue-gel loading buffer to each of your tubes. (Add 4.0ul of tube G to each of tube F and tube B). Your digests are now ready for gel loading.

4. Prepare the agarose gel electrophoresis apparatus. Make sure the comb is situated closest to the negative (black) electrode. You will be shown a demo so please take good notes at this point in time.

5. Take your premelted agarose solution (tube H), and slowly pour into your electrophoresis gel bed up to the level of the sides of the gel plate. Allow the gel to set for 30 to 45min.

6. When the gel has set, you are now ready to load your samples. Remove the ends of the gel plate carefully without disturbing the gel. Also remove the comb gently by wiggling it up without tearing the gel.

7. Slowly dispense all of the blue TBE buffer (bottle J) into the gel chamber. The liquid should cover the surface of the gel.

8. Load the samples into the wells of the gel using a P20 set to 20ul as follows (the loading technique will also be demonstrated):
(note, it doesn't really matter what order you load your samples as long as you know the order that was loaded).
Well #1 Load 20ul of tube B sample (Lambda + no enzyme, negative control)
Well #2 " " tube C sample (Lambda precut with HindIII, positive control)
Well #3 " " tube D sample (Lambda precut with EcoRI)
Well #4 " " tube F sample (Your digest, Lambda cut with HindIII)

9. Close the lid of the gel chamber. Connect the power cords. Turn power supply on and set to 75volts. The gel should take about 2 hours to run. Remember, that the gel should not be handled at all unless power has been turned off.

10. After approximately 2hrs, your gel should be ready. You can use the mobility of the blue dye as an indictor of how far the gel has run. In general, you want to run the gel until the blue dye has reached the bottom of the gel.

11. Remove the gel carefully (it is quite delicate and will break if not kept flat), and place into plastic container.

12. Pour "Final Carolina Blu Stain" (bottle M) into plastic container to a volume just enough to cover the gel. Incubate for a minimum of 15min with gentle agitation.

13. While gently pressing the gel against the bottom of the container to prevent it from sliding off, pour the staining solution back into bottle M for reuse.

14. Destain the gel by covering the gel with deionized water (bottle K), agitate for 15minutes. Pour stained water down the sink. You will probably need to repeat this step at least two more times. Hopefully, you will see distinct bands being more and more clear as the stain is being washed away.

Restriction Digests and Gel Electrophoresis:  some notes.

READ THIS FIRST...
O.K. this is probably the most complicated of the kits we have to offer, so it's extremely important that you are on top of the game when you do this with your class.  Firstly, a little background.

FIRST thing you're going to do is a restriction endonuclease digest (or "digest" for short).  This is simply taking your DNA and cutting it up using an enzyme that recognizes a very specific sequence.  For instance, we are using HindIII in our digestion and this particular enzyme recognizes the following:

So,.. you have to picture this enzyme bumping on and off your piece of DNA, and everytime it sees this sequence, it will cut it. 

You are cutting a pretty big piece of DNA called lambda DNA.  It's actually an entire genome from an organism commonly called a bacteriophage which is really science-speak for a virus that infects a bacteria.  This piece of DNA is 49,000base pairs (bp) long or 49kilobases (kb).  Since people already know the exact sequence of this piece of DNA, we can tell you that the HindIII recognition sites happens at 7 places along this 49kb stretch.  If you do the math, that means you should get 8 fragments and I'll tell you the sizes of each one a little later.

The procedure itself is fairly straight forward... Really all you need is the enzyme and the DNA to be cut.  Both of these components will be floating around in a buffer that is essentially designed to keep the enzyme happy.  Your buffer in this case is tube E.

You will want to incubate this digest at around 37C for a minimum of 1 hour.  Most scientists will routinely let it incubate overnight if you want to make sure that the digest is complete.  If you don't have time to set up the gel stuff at this point, you should put your digests (after incubation) in the freezer until you need them.

SECOND thing you need to worry about is the preparation of the agarose gel.  This stuff is supplied to you in big glass bottles.  You will need a microwave to do this easily.  Before you place your agarose bottle in the microwave, untighten the lid of the bottle so that no pressure builds up when your agarose is boiling.

N u k e   the agarose until it starts to boil (bubbles!).  Take out your bottle and give it a bit of a swirl (be careful, it will be hot).  Put it back in the microwave for another 30 seconds or so.  Give it another mix, and take a good look at the dissolved solution.

 WHAT ARE YOU LOOKING FOR?
You are looking for "chunkies."  You don't want "chunkies";  you want your agarose completely dissolved so that your DNA runs smoothly through the gel.  This is where things may be a bit tricky because you want to make sure that everything is dissolved, but at the same time you don't want to overboil in case too much liquid evaporates away!

AT THIS POINT, you'll need to set up your gel apparatus.  There should be a plastic contraption (see figure 1) with notches to fit combs.  Follow the figure and make sure you seal the edges well with some good tape.  Once this is set up, your gel will hopefully be ready.

You may be lucky enough to receive a newer gel setup. If so, it's actually much easier to set up (no tape). Instead, there are "dams" which are these black metal like contraptions. Follow the picture below for set-up, and then proceed with the gel pouring.

When the gel has cooled down (to about 60C), add about 10 drops of carolina blue stain (This is actually optional). Mix thoroughly and then carefully pour the gel mixture (now tinted blue) in the plastic apparatus you've just set up.  If you see bubbles, near the wells just use something to "move" them towards the side of the gel.  Now, you will want to wait for your gel to solidify... This will take about 30 minutes.  If you're in a hurry, you can put it in the fridge.

O.K. , when your gel has solidified, you can put the whole thing inside the gel box (remember - DNA is negatively charged and needs to RUN towards the positive electrode RED RED RED IS POSITIVE ). Don't forget to remove the combs and tape!!

            You should have about 2 litres of 1X TBE buffer.  This is your running buffer which contains ions that allow electricity to be conducted throughout the gel.  You will also need to add some carolina blue stain (about 4.0mls - although this is actually optional).  Pour the TBE buffer into the gel box set up, until the agarose gel is covered (maybe about 2-4mm of liquid covering the gel).

Your gel is now ready for samples to be loaded.  AT THIS POINT EACH GROUP WILL HAVE 4 samples to load:

• F:        This is your test digest.  You added l DNA, buffer, and HindIII.  You will need to add 4.0ul of the loading dye (tube G) before you load it into the gel.
  
• B:        This is a negative control. It is identical to tube F, except it has no HindIII enzyme.  You will also need to add 4.0ul of the loading dye (tube G) before you load it into the gel.
  
• C:        This is a positive control.   This is exactly the same as tube F, except that we have prepared it for you and checked it out before.  You have this to compare with your test digest. It will already have loading dye in it.
  
• D:        This is l DNA cut with a different restriction enzyme. Basically, this is just a sample for comparison. 
  This will also already have loading dye in it.  Here l DNA was cut with the enzyme EcoRI.  EcoRI recognizes the following sequence...
  

You may have noticed that you have two rows of wells.  This means that you will load two lanes of samples and run the samples only through half the gel.  After loading the samples (you'll want to load 20ul of each sample), you should connect all the plugs and turn the power pack on to high.

            Make sure all the connections are colour coordinated.  Actually, just make sure your DNA will run towards the red electrode (red is normally positive).  The power pack set to high should give you a setting of about 115V.  Since the gel is quite large, the gel will take considerably longer to run.  Run your samples until the dye front (the blue stuff) travels half the gel.  (May take between 2 - 6 hrs).

WHEN the gel has finished running, you will need to find a tupperware container or dish that can hold the gel.  Carefully place the gel in this container and add the stain solution (the M bottles ) until the gel is covered.  Although the original protocol states to stain for 15mins with gentle agitation, I would highly recommend staining for about an hour.  This is largely because the gel is much bigger and may need more time to stain properly.

            The destain steps should also be lengthened to about an hour as well.  These "detain" steps you repeat until you feel your bands are easy to visualize.  If time isn't a factor, it doesn't hurt to have an overnight destain step.  If you find that you are running out of distilled water for the destaining, you can use BOILED bottled water or even BOILED tap water in a bind.

O.K. What should your gel look like...
Hopefully it looks a little like this:

These numbers have been calculated because the sequence for lambda DNA (all 49000bps!!) has been figured out.  Since we also know precisely what sequences the restriction enzymes cut, we can easily predict the fragment sizes.

            ESSENTIALLY, you're looking for the following things.  Firstly your tube B is an uncut control.  Here you didn't add an enzyme so your DNA should stay big.  Consequently, you will hopefully see one band (might be a bit smeary) near the top of the gel.      
The other thing you're looking at is your tube F.  This was your digest.  In theory it should
look like tube C (positve control) if everything worked o.k. 

ALRIGHT THEN... I'll leave the gel explanation to you, but you're welcome to use my "ball of string and hamster" analogy.