Wednesday, May 1, 2013

Primer Design Cont.

I am continuing to design probes for In Situ Hybridization. Currently, I used Ensembl and BLAST to find gene orthologues in the Tiger Salamander (Ambystoma).  See Making Primers Part 1 for reference. After I received the primers, I tested them out via a PCR reaction and my cDNA template (generated kindly by our undergrad Minami Tokuyama). 7 out of 10 of the primers seem to work well. This is continuation of the blog Primer Testing and Running a Gel. Note that this article is not peer reviewed and is subject to change.

Now that I have my gel, I can begin to analyze the results. 3 of the expected bands do not show up. The other 7 look good, as there are no strong multiple bands that would indicate non-specific binding. The 100 bp ladder can be read from the bottom up, with the first band in the first well as 100 bp, then next as 200 bp and so on.

Recall that small sequences move more quickly during gel electrophoresis, and should travel much farther from their starting point (the top of the gel) towards the bottom. Large sequences are slow and won't move as far. Therefore, the 5th band from the bottom of 100 bp DNA ladder, which is much brighter, indicates PCR products that are roughly 500 base pairs. I can see that seven of my gene products are between 400 and 600 basepairs. 

This is good news to me, as this was the expected outcome.

I consult my spreadsheet to see what the predicted PCR product sizes are. I had previously determined this information by entering my primer sequences (forward and reverse) into the Sequence Manipulation suite: PCR Test, at

I can see that in well 2, which is pax6 (exon2, 2nd primer set), should be 404 base pairs. I can see that in well 4, mef2a (exon1, 2nd primer set) should be the largest at 546 base pairs. Indeed, it is the largest sequence and has moved the slowest (from top to bottom, or from the black negative anode to red positive cathode). Most of the products should be around roughly 450 bp and this is what my gel reflects.

I can see faint Primer Dimer bands which are located under the 100 bp band (towards the bottom). This doesn't prevent me from using the primers; however, bands over 100 bp are troublesome as they might indicate the primers are binding nonspecifically. 

After the primers have been tested and deemed correct, based on predicted PCR product size, reorder the primer with T7.

Primer Testing and Running a Gel


The primers will come dry, in small tubes. They are stable at room temperature. As soon as you add water, you will want to keep the primers on ice. You will need to make a 200 millimollar stock of the primers. I also recommend making a working stock as well.

To make the 200 mm stock, find the volume in nanomoles and multiply by 5.

Primer stock

For example:

Gene1-1F   -  29.4 nm  x 5 = 147 ul
Gene1-1R  -  35.1 nm  x 5 = 175.5 ul
Gene2-1F  -  29.6 nm  x 5 = 148 ul
Gene2-1R  -  29.2 nm  x 5 = 146 ul

Take that amount of microliters per tube and add Molecular Grade ddH20 to the tube. You now have a 200 mm stock.

Vortex the tube to mix them. Spin them down in the centrifuge. 

Working Stock

For regular PCR, the concentration should be at 20 mm. For RT-PCR, you want it to be at 10 mm.
Using the C1V1=C2V2 equation  :   (200 mM) x = (20 mM) (100 ul)
                                                                               x = 10 ul

You want a working stock of 100 microliters. You can put both the forward and reverse primer into your working stock. So 10 ul/2  = 5 ul of the forward primer and 5 ul of the reverse primer.

Add 90 ul of Molecular grade water
          5 ul of forward primer
          5 ul of reverse primer
Total of 100 ul working stock

Vortex the tube to mix them. Spin them down in the centrifuge. 

You want to keep both the long-term 20 mm stock and the working stock (F+R primers) on ice, or in that -20 to -30 C freezer.


Next, you want to create a Master Mix solution. You want the number of reactions + 10% room for error. Total volume for each well (each primer being tested) is 20 ul. If you are testing 5 genes, then that would be 5 reactions (or 5 wells). So take the Mastermix recipe and times it by number of reactions and 1.1 for each of the elements.

Do not add the primer working stocks to the Mastermix. If you are using different templates, say cDNA from mouse and cDNA from frog. DO NOT ADD IN THE TEMPLATE yet to the Mastermix. However, if you are testing genes for the same species, with the same template you can add the template to the Mastermix. Let's assume that all the 5 genes I am testing are for the same species. I will add 22 ul of template to the Mastermix solution.

Next, get some tiny PCR tubes (and lids). Remember, the desired volume for each PCR tube is 20 ul (For regular PCR. For RT-PCR, you want 10 mm). You want to add 19.2 ul of Mastermix to each tube. Then you want to add 0.8 ul of each primer working stock (changing pipette tips each time!!). Then you total volume is 20 ul.

Each PCR machine varies, but here is the general procedure:
Put the tubes in. Tighten the lid.

-20 ul volume
-Heated lid
-Last step should run at 4C forever.

Check with your lab about the specific program you want to run (with temps, times, and cycles). Our PCR machine has a program pre-set.

Here is the program I use for PCR.

5 minutes         94 degrees      (x 1 cycle)
1 minute          94 degrees      (x 40 cycles)
1 minute          55 degrees      (x 40 cycles)
2 minutes        72 degrees      (x 40 cycles)
7 minutes        72 degrees      (x 1 cycle)
15 minutes      4 degrees        (x 1 cycle)
(set on 4 degrees forever, if you want to leave it overnight)

If you want to know what occurs during each step (of the denaturation, annealing, and extension process), check out NCBI's explanation.


Right before you want to run the gel, add a loading dye to each well. I use a 6x loading dye, but it needs to be at 1x. I use the C1V1=C2V2 formula to solve for x:

                              (6x)(y) = (20 ul)(1x)
                                       y = 3.333 ul

I want to add 3.3 ul of 6x loading dye to each tube. This will turn the solution from clear to blue. Spin the tubes down.

Mastermix + Primer and Templates in PCR reaction tubes


First you want to make the 1-2% agarose gel.

Depending on your gel casting tray size, you will need different volumes. I am running a small gel and my casting tray holds about 75 ml. To test how much volume it holds, just pour water in from a graduated cylinder to find out. I am going to make slightly more than I need to be safe. A clamp is used on the gel casting tray. Put the comb in. This will create wells when you pour the agarose in.

Gel Casting tray

If I want 82 mls of agarose gel mix, I will use 0.82 grams of agarose powder.

I add the powder to 82 mls of Borax.  Next I want to microwave it, until it boils. DON'T LET IT BOIL OVER! You may have to stop and wait a few times, until all the particles are dissolved. The solution below needs to be microwaved longer.

1% Agarose Gel solution

You want the solution to be clear.

When the solution is clear, wait for the agarose gel solution to cool down. When it is room temp, you want to add Ethidium.

Ethidium is a dangerous chemical and is usually kept in a safe place, stored at room temperature. For a small gel, use 1-2 ul of Ethidium. For a large gel, use 5 ul. In our lab, we use a pipetter reserved for the Ethidium only and kept in a cleared out area assumed to be ethidium-dirty.

Swirl the bottle to mix up the Ethidium into the agarose gel. Pour the gel in the gel casting tray, as pictured above. It should be 15 minutes or longer for the gel to congeal. If you wait too long it will solidify in the bottle.


When the gel is solid, you can unclamp the casting tray and carefully remove the tray with the agarose gel in it. It should be rectangular and jelly-like.

Move the gel to the gel electroporesis box.

Pour in Borax to cover the tray. Don't fill it past the MAX line.

Pull the comb out. If you place a dark sheet under the box, you should be able to see square wells. This is where you will pipette your mix into.

For a large gel, you want to put 8-10 ul of each Mastermix+Template+primers+loading dye into each well. For smaller gels, 2-4 ul might be adequate. If you run your gel and get blobs and trails, it may be because there is too much DNA. Cutting the concentration will help.
Use a different pipette tip each time!

In the peripheral wells, you want to use a DNA ladder. I use a 100 bp ladder and a 1000 bp ladder. This will allow you to gauge how large your product is compared to a reliable measure of size.
In general, you can use about 5 microliters for the 100 bp ladder and 2-3 microliters for the 1000 bp ladder. 

Wells are filled in the gel electroporesis box

Write down which primer is in each well!

Put the cover onto the electroporesis, matching black to black and red to red.

Turn the gel electroporesis machine on. I use a setting of 110, then start it.

You should see bubbles coming out of the anodes. Let your gel run for 20-45 minutes. You want them to run down 75% the length of your gel. If you let it run too long, it will run right off your gel!

A finished gel should look like the one below:

Note: I disposed of my gel in a special hazardous waste ethidium bromide container.

Next, I photograph the gel using a special Gel-Doc machine that uses Trans-UV to image. I invert the image when exporting as a .TIFF file to get the picture below:


Gel Doc

Recall that bigger fragments move more slowly and smaller fragments move faster. To give you an idea of relative size, the DNA ladders (100 bp and 1000 bp) are used. Since the fragments will run from the negative electrode (black) to the positive electrode (red), the slower fragments will be closer to the starting point. Read the ladder from the bottom up. See below. The brighter bands are 500 and 1000.

You want your band to be a single, bold band. Multiple bands can indicate contamination or non-specific binding of your primers.

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