WET LAB DNA Barcoding: From Samples to Sequences - Northwest ...

WET LAB
CLASS SET
11. To prepare your DNA (PCR) sample for
the gel (see Figure 3.3):
a. Label a new 1.7 ml microfuge tube
with your name and the word “gel.”
b. Add 5.0 μl of 3X loading dye
(labeled “3X”) to your new “gel”
microfuge tube.
c. Add 10.0 μl of your PCR sample to
your “gel” microfuge tube.
Transfer Transfer your your 50 50 µl µl PCR PCR to
to new a New Microfuge Tube Tube
Labeled with Your Name
and “PCR”
PCR
Transfer your 10 µl 10 of µl your of your PCR
PCR to to a New new Microfuge Tube
Labeled with Your Name
and “Gel”
Figure 3.3: Transferring Your PCR Reaction to Microfuge Tubes.
Gel
12. Have a member of your group prepare the PCR Negative Control (tube “0”) for the gel:
a. Label another new 1.7 ml microfuge tube with your group’s name, “negative control,” and “gel.”
b. Add 5.0 μl of 3X loading dye (labeled “3X”) to the negative control “gel” microfuge tube.
c. Add 10.0 μl of the negative control PCR sample “0” to the negative control “gel” microfuge tube.
13. Cap the “gel” microfuge tubes, and mix them by either flicking the tube with your finger and tapping the
tube on your bench top, or by vortexing and then spinning the tubes in a microcentrifuge.
14. In your lab notebook or on a separate sheet of paper, make a table similar to Table 3.1 to list where you
will load each sample from your group on your DNA gel. You will also be loading molecular weight standards
on each gel. Molecular weight standards (sometimes abbreviated “MW”) contain pieces of DNA of known
sizes (see Figure 3.5 at the end of this handout). This will help you determine whether your PCR reactions
produce DNA bands of the expected size (about 650 base pairs). Be sure to include your molecular weight
standards in your table and on your gel!
[Note: The Well Number is the slot on the
gel in which you will load your samples
(see Figure 3.4). Most gels have 8 wells,
but some have only 6, and others have up
to 20. Check to see how many wells are
in your gel.]
15. Put your gel in your gel box and
add enough DNA Gel Buffer to the
gel box to just cover your gel.
Remember: “Run Towards Red.”
Be sure that the top of your DNA
gel (where the wells are) is near the
negative, or black, electrode on the
gel box, and the bottom of your gel
is near the positive, or red, electrode.
That way the negatively-charged
DNA will always “run towards red.”
Well Number Sample Name
1 ________________________________________
2 ________________________________________
3 ________________________________________
4 ________________________________________
5 ________________________________________
6 ________________________________________
7 ________________________________________
8 ________________________________________
Example:
1 ________________________________________
Molecular Weight Standard
2 ________________________________________
Joe Ting’s Salmon PCR-1
Table 3.1: Samples Loaded onto DNA Gel
Wet Lab – DNA Barcoding: From Samples to Sequences
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©Northwest Association for Biomedical Research—Updated October 2012