For this experiment you will purify the digested yeast genomic DNA and pBS plasmid DNA using agarose gel electrophoresis. You will physically isolate the linear plasmid DNA and yeast genomic DNA from the 0.5 to 1.0 kb size interval using a razor blade to excise the relevant agarose slice from the gel, and then purify the DNA via spin columns and precipitation in EtOH. (Note: Nucleic acids such as DNA and RNA are insoluble in high concentrations of EtOH.) You will verify the yield of your DNA recovery via a 2nd agarose gel and set up a DNA ligation reaction to covalently join the yeast "insert" and pBS vector DNAs.

1) Pour a 0.7% agarose gel - use large toothed comb.

2) Thaw restriction digests and add loading dye.

3) Load and run digestion samples on the agarose gel.

4) Excise the linearized pBS DNA, which is 3.0 kb, and yeast genomic DNA in the 0.5 to 1.0 kb size range.

5) Isolate the DNA from the gel slices using spin columns and precipitate the DNA in EtOH.

6) Pour an agarose gel using small toothed comb.

7) Spin down the DNA in the micro-centrifuge. Wash the pellet with 70% EtOH, dry in the SpeedVac, and resuspend the DNA in TE buffer.

8) Run out a small aliquot of the purified DNAs on an agarose gel.

9) Set up DNA ligations. Incubate overnight at room temperature.

The agarose used in this course is of sufficient purity that DNA can be extracted from it via a variety of means and then be used in subsequent enzymatic reactions (e.g. restriction enzyme digestions, ligations, etc. ). Agarose gel electrophoresis will allow you to separate the two digested pBS fragments (what are they?) and the many (approximately how many?) yeast genomic DNA fragments derived from the restriction enzyme digestions of last week. There are a wide range of techniques used to then isolate and purify the DNA from the gel. We will be using one particularly high tech method which relies on a razor blade, a hypodermic needle, and a little bit of glass wool (see attached protocol). The DNA in TAE buffer that is eluted from the gel slice will then be further purified via precipitation in EtOH. For this and subsequent EtOH precipitations, a good rule of thumb is to add 1/10 volume (compared to the starting volume of DNA) of 3M NaOAC (pH 6.8), and at least 2.5 volumes of 95% EtOH. Mixing and incubating this solution in the freezer or on dry ice will quickly cause the DNA to precipitate out of solution. It can then be collected via centrifugation.

It is generally useful to measure the efficiency of your recovery of DNA from the agarose slice. If things work well, you should recover ~50-80% of the initial DNA from the gel. If things don’t work so well, this figure can easily drop to 0%. This will help us decide how much of your DNA to add in the subsequent ligation reaction.

Both the plasmid DNA and the insert DNA have been digested with a two restriction endonucleases, Eco RI and Bam HI. Both these enzymes cleave phosphodiester linkages in double stranded DNA at specific recognition sequences, GAATTC for Eco RI and GGATCC for Bam HI. What do you notice about these recognition sequences (hint: write out the complementary strands)?

Shown below are the products from an Eco RI digestion.

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The Eco RI enzyme cuts after the G residue, leaving what is referred to as a 4 base 'sticky end'. This 'sticky end' can base pair with the complementary sticky end generated by any Eco RI cleaved DNA fragment. In this way one can join together Eco RI generated restriction fragments from any DNA sources. The same holds true for DNAs digested with Bam HI.

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In our experiment, when two such fragments form transient duplexes, the enzyme DNA ligase joins the two molecules via formation of a covalent phosphodiester linkage. Significantly, any two DNA molecules that contain compatible sticky ends can be joined together via ligase. This makes it is possible to generate recombinant plasmid constructs containing heterologous animal or plant insert DNAs.

1) Pour a 0.7% agarose gel as described in last week’s protocol.

2) Thaw restriction digestion samples from last week. Add 5 m l of 10 X DNA loading dye to each sample. Mix and spin down briefly (5 seconds) in micro-centrifuge to collect all liquid at the bottom of the eppendorf tube. Be sure to also set up a tube of DNA molecular weight markers.

3) When the gel has solidified, load samples into 1-2 wells each.

Run out samples at 100V.

4) While the gel is running, use a sterile 26 gauge hypodermic needle and poke a small hole in the bottom of 2 small 600 m l eppendorf tubes. Remove the lids of these small tubes with a razor or scissors. Label these tubes "insert" and "vector".

5) Place a small plug of aquarium filter floss in the bottom of the small eppendorf tube.

6) Stop the electrophoresis and take a picture of the gel. You should see one strong DNA band for the pBS sample and a smear for the yeast genomic DNA (why?).

7) While placed on the illuminator, take a sterile razor blade and excise a band of agarose that contains the pBS vector band (3.0 kb) and insert DNA (from the 0.5 - 1.0 kb range). Place these slices into the appropriately labeled 600 m l eppendorf tubes.

8) Place the two small eppendorf tubes into 1.5 ml eppendorf tubes.

9) Centrifuge these tubes for 5 minutes at 5000 rpm in the micro-centrifuge. Liquid containing the DNA will flow from the agarose slice into the larger eppendorf tube.

10) Estimate the volume of liquid recovered, using a P-200 pipetman. Transfer the liquid into a new 1.5 ml eppendorf tube.

11) Add 1/10 volume of 3M NaOAc and >3 volumes of 95% ethanol (you can fill the tube to the top if desired). Invert tube to mix and place in crushed dry ice for 10 minutes.

12) Spin tubes for 10 minutes in the micro-centrifuge to pellet the DNA. BE SURE TO PLACE THE TUBES SO THAT THE HINGE IS FACING OUTWARD- THIS WILL ALLOW YOU TO MORE EASILY FIND YOUR DNA PELLET AFTER SPINNING.

13) Remove tubes from micro-centrifuge. Hopefully you will see a small whitish pellet at the bottom of the tube on the side with the hinge.

14) Carefully remove the EtOH using a P-1000. Be careful not to disturb the pellet. It is all right if there is still some residual EtOH in the tube at this stage.

15) Gently rinse this pellet with 500 m l of 70% EtOH. Be careful as the pellet may come loose with the 70% EtOH wash. If the pellet does dislodge, briefly spin the sample down in the micro-centrifuge for 1 minute. Remove the 70% EtOH using a P-200 pipetman. This step helps to remove salt from the pellet.

16) Remove the tubes from the micro-centrifuge and carefully remove the 70% EtOH using a P-200 pipetman. The pellet may not be as easily seen as it previously was, so be very careful. Remove as much EtOH as possible using the pipetman.

17) Dry the pellet in the SpeedVac for 2 minutes or until tube is dry. Be sure that the rotor is spinning before you pull down a vacuum.

18) Resuspend the pBS DNA pellet in 10 µl of dH2O. Be sure to vortex the tube so that the pellet is completely dissolved.

19) Resuspend the yeast genomic DNA pellet in 10 µl of dH2O.

20) Pour a THIN 0.7% agarose gel with small tooth comb. Use only 50 mls of agarose solution.

1) Label 2 eppendorf tubes:

2) Set up the ligation reactions as follows:

Tube 1 Tube 2

buffer

3) Incubate samples at room temperature overnight.

4) Add 9 µl of dH2O and 1 µl of 10X DNA loading dye to remaining pBS and yeast DNA tubes. Load samples along with DNA molecular weight markers on agarose gel. Run gel for 15 minutes at 100 Volts.

5) Stop gel and take picture. Hopefully you will see the DNA recovered from the agarose gel. This will help you to interpret the results of your transformation experiment next week.