Frequently Asked Questions

BASICS

1. What is the best way to get started?

a. Step-by-step online tutorials
b. Help Tab within Visual OMP
c. Within any page in VO, press F1 key to see context-dependent help files
d. Go to Training Materials

2. How do I maximize virtual memory?

On a Windows XP system, goto System Properties, Advanced, Performance, Change Virtual Memory (usually can change to 4096 max is disk space is available)

3. What are the “allowed” characters in the oligo name box?

As far as characters go, users are only able to type in the following:
"A" To "Z", "a" To "z", "0" To "9", "'", "@", "&", "^", "$", "!", "-", "#", "%", "{", "}", "~", "(", ")", "_"
These are the legal DOS file name characters. You may be able to import in “.” and “blanks”, but it may produce sporadic results. For example, and 2 oligos named “oligo 1” and “oligo_1” would result in the same graphic for both oligos.

4. I have a European operating system so I use commas instead of dots – will this work?

No. If the user is writing 0,05 M salt instead of 0.05 M salt, the oef will read it as 5 M salt.
We are currently working on an improvement so our European users will be able to use commas.

SIMULATION

5. How does effective Tm change with target and probe/primer concentration?

See Tips and Techniques #19

6. How do I import in a series of sequences?

If your list of oligos is in a FASTA file, you can go to “File”, select “Import” and browse for your .fas file. Oligo names and sequences will be imported into your experiment. CAUTION: Please only use legal dos characters in your oligo names. See FAQ#3.

7. How do I see the list of bases from the graphic view?

Right-clicking and choosing “View Source” on the graphic will show you the list of base pairs.

8. What are the differences of percent bound and percent bound (2)?

The first percent bound column reflects the % bound of the first species listed in your name column (i.e. first species is “Target” in Name = ”Target+probe1”) to your second species (probe1). The second percent bound column is the % of the second species bound to the first species (here, it is % of probe1 bound to target).

9. What is custom Tm?

The definition of Tm = concentration of intended species (het) < ½ (limiting strand, usually target). However, if you want to see the temperature at which the heterodimer concentration is more than ½ (or 50%), you can also specify this percentage to a custom one. For example, a custom Tm at 75 will find temperature at which concentration of intended species (het) < ¾ of (limiting strand, usually target).

10. If I am importing in 100s of probes, how do I change a column (e.g. Concentration or Function), all to the same description (i.e. fixed probe)?

a. Export your Sequences Tab Page in VO5 to Excel.
b. Under function on the first sequence row, write in Fixed Probe.
c. Select this Cell.
d. Left-click on the right-bottom corner of this cell and keep mouse button down.
e. Drag the box down with the mouse until all 100 rows are selected.
f. Right-click and select “Copy cells”.

Now all of your probes should be “Fixed Probe”
g. Copy the total function-column (all 100 saying Fixed Probe).
h. Go into the Sequences Tab in VO5.
i. Select the function cell of the first sequence row.
j. Right click.
k. Select “Paste cells”.

DESIGN

11. How do I design the best primers/probe for a larger DNA target?

a. Seq Tab: Input sequence, concentration, double or single stranded, function=target,
Description=accession number
b. Settings Tab: Input in temp, Na and Mg conc,
c. Simulate target alone
Under Design Settings - check Blast Designs and search through local FASTA file containing your target labelled with the accession number indicated in the Seq Tab.
c. Number of solutions = 50 is usually a good place to start.
d. Designs Tab: Input in target sequence, select target position
“Select from Display”: choose a region with little secondary structure
Examples of target position:
400-500: probe sits in between 400 - 500
135: probe covers SNP135
100-150: primer pairs flank nt 100-150
e. After the design is done, copy the desired probe/primer (usually solution #1) back into experiment (right-click and select "Add to experiment").
f. Simulate to check percent bound and eff Tm with target (whole or subsection)

12. What are the exact positions of these primers?

See Tips and Techniques #8

13. Why am I not getting any design results?

View Design information, and relax those parameters which affect the reasons why the design failed. For example, if most candidates failed mishyb, go back to adv parameters and increase the mishyb Tm and lower the mishyb dG.

14. What is the process of primer/probe selection?

Candidate Generation based on maxpolyn, GC content, naïve Tm
CANDIDATES
Pick n = Num_red x num return (number of solutions in the settings tab)
NOMINATIONS
Filter 1: Oligo monomer Tm and dG, Oligo homodimer Tm and dG, Tgt hairpin
N2 = Num_red x 2
QUALIFIED NOMINATIONS
Filter 2: Mishyb Tm, dG
SELECTIONS
Filter 3: Crosshyb Tm, dG
SOLUTIONS N3=num return=# solutions