During the gel purification we are going to load the PCR fragment and the digested vector
on an agarose gel, apply electric current, and separate DNA molecules based on their
molecular weight.
Then we will cut out DNA bands from the agarose and clean them with a gel purification kit.
Gel purification allows us to separate vector DNA from uncut fraction, clean PCR fragment
from unspecific PCR products and primers, and also serves as a quality control for the
molecular weight of vector and insert.
The summary of gel purification protocol is presented here. Please refer to the Protocols
poster for additional details.
Gel purification protocol:
Prepare 1% agarose gel Add 1 µg of agarose to 100 ml of TEA buffer
Boil in a microwave until the agarose completely dissolved
Add 2 µl ethidium bromide or GelRedTM Pour into a casting tray
Leave for 30 min to solidify
Load the gel Molecular weight marker
1 µl of undigested vector Vector restriction digest
Insert restriction digest
Run the gel until bromophenol blue dye reaches the end
Cut out desired DNA fragments
Purify with a gel purification kit
The advantage of doing vector restriction digest in parallel with PCR is that they both
are ready about the same time for gel purification. This way we can run both vector and insert
on the same agarose gel.
Let's prepare a 1% agarose gel. I am going to weigh 1.5 grams of agarose and add it
to 150 ml of TAE buffer. Then I heat it up in a microwave until
it boils.
A safety note: please wear heat-protective gloves when you handle a bottle with boiling
agarose. Leave the screw cap on the bottle somewhat loose to allow for vapours to vent.
Then I let the agarose to cool down for 15 min. Add ethidium bromide. Swirl the bottle
to mix up ethidium bromide.
Another safety note: remember that ethidium bromide is a cancerogen and change your gloves
each time when you get in contact with gels, trays or any other contaminated equipment.
Now I am going to pour the agarose in a tray with a comb
and let it solidify for 30 minutes. Finally, I am going to transfer the gel into
a running tray and filled with the TAE buffer.
By now both vector restriction digest and the PCR are ready. So, on the agarose gel
I am going to run:
Molecular weight marker 1 µl of uncut vector
entire vector restriction digest and all 50 µl of PCR reaction
To each of these three tubes (except the marker) I have already added 5 ul of the loading dye,
which contains bromophenol blue and glycerol.
I am back to the gel room with my tubes and I am ready to load the gel. Once again: molecular
weight marker… uncut plasmid… cut plasmid… and the
PCR product.
I will run the gel for 35 min at 105 V. Remember, that DNA is negatively charged and therefore
it will run towards the positive, or red electrode.
The run is over, let's take the gel to a UV transilluminator and have a look at it.
Here is our DNA separated on the agarose gel. Let me put some labels here. In the left lane
we have the marker, then uncut vector, cut vector and the PCR product.
There are few pieces of information that I extract from this kind of picture. First of
all, I compare uncut and cut vector lanes. You see how the entire vector band shifted.
This means that the vector was linearized.
I also look at the molecular weight of the linearized vector and compare it with the
information from the sequence file. The size ofpSAT6-MCS is supposed to be 3900 base pairs,
which is in agreement with what I see on the gel.
In addition I roughly estimate the amount of vector DNA. The rule of thumb is that if
you can see it well on the gel, then you should have sufficient amount for efficient ligation.
In the case of the PCR product I also look at the molecular weight and at the amount
of DNA. Right now everything looks great, so I will
proceed with cutting out the vector and the PCR fragment from the gel.
Before cutting the gel I prepare two labelled Eppendorf tubes, two razor blades, and a UV
shield. Now I am going to put the UV shield on, pull
out the transilluminator from the imaging system and turn on UV light.
Cutting gels is pretty straightforward. I usually cut off gel to the right of my band,
then to the left, then top and bottom. Then I pick a gel fragment with a razor blade and
transfer it to a labelled tube. The same about the PCR band. I cut it out
and transfer to an Eppendorf tube.
Now I am going to use a gel purification kit to extract DNA from the gel. If you have used
gel purification kits before, fast forward to Step 5.
The GFX gel band purification kit I have here on the bench comes from GE Healthcare. It
consists of GFX DNA binding columns, a capture buffer, and a wash buffer with ethanol.
I start gel purification by weighing my gel bands. Then I add equal amount of capture
buffer. To dissolve the agarose I incubate the tubes at 65 oC for 10 min. Then I transfer
the solutions with dissolved agarose to the GFX columns. I spin the columns for 30 seconds
in a microcentrifuge.
Then I add 500 µl of wash buffer. Spin the columns again. Then I place the columns into
new tubes and elute DNA by adding 50 µl of double distilled water. And doing one last
spin in the centrifuge.
At the end of this step our vector is cut, cleaned and ready for ligation. The PCR product
still have to be cut with XhoI and BamHI.