Frequently Asked Questions

1. What if my DNA concentration is too low? If your DNA concentration is lower than what we recommend on our website please be sure that you bring it up to the proper concentration before submitting for sequencing. There are several methods you may consider for concentrating your DNA including the use of a speed vac or ethanol precipitation. Please be advised that the success of your sequencing reaction is heavily dependent on the purity and concentration of your DNA so please adjust your protocols and concentrations accordingly.

2. What is the current turnaround time? Our turnaround time is approximately 1-2 business days but we are frequently able to give 24-hour turn-around M-Th. Samples are sequenced on a first come first served basis so turnaround time can vary depending on current workload, when you submit your samples, and how many samples you have. We typically start setting up sequencing reactions by noon so submitting your samples in the morning will give you the quickest turnaround time.

3. Which universal primers does the UCDNA Sequencing Facility provide? We provide the following universal primers free of charge: M13-21, M13-R, T7Promotor, T7Terminator, T3, and SP6.

4. How does cycle sequencing differ from PCR? Cycle sequencing is a much more stringent protocol than PCR in that it uses only one primer for extension and therefore is a linear extension rather than an exponential extension. This means that the growth of the product is linear and not exponential as in a PCR reaction.

5. If my primer worked for PCR why didn’t it work for sequencing? Again, because sequencing is a linear amplification and more stringent than PCR, if the primer does not anneal very efficiently and accurately, you could end up with either very weak and noisy data or, in some cases, no data at all. In a PCR reaction, there are two primers at work generating a product that grows exponentially so even if the primer/primers are not very efficient there are many more opportunities for the reaction to “go” and you should still end up with a product. So, be forewarned, even if the band looks great on your gel, your PCR product might not sequence well, if at all!

6. How do I interpret my sequence data? First and foremost, even the best sequence data can contain up to a 2% error rate so you must look at the electropherogram for your sequence! You are responsible for manual editing of your sequences so accurate sequence data can only be determined from the electropherogram. This is critically important to the integrity and validity of your sequence! Please do not rely on your text file. Visit our software downloads page for freeware sequence viewing software if needed.

7. How do I interpret the ambiguous bases in my data? The letters used for ambiguities are called IUB codes. The IUPAC-IUB codes are used to define multiple nucleotide possibilities in one position. The table below indicates the definitions:

Code

Represents

Complement

A

Adenine

T

G

Guanine

C

C

Cytosine

G

T

Thymine

A

Y

Pyrimidine (C or T)

R

R

Purine (A or G)

Y

W

weak (A or T)

W

S

strong (G or C)

S

K

keto (T or G)

M

M

amino (C or A)

K

D

A, G, T (not C)

H

V

A, C, G (not T)

B

H

A, C, T (not G)

D

B

C, G, T (not A)

V

X/N

any base

X/N

-

Gap

-

8. What if I want a sequence run again? If your sequence fails due to an error on our part, we will automatically re-run the sequence free of charge. However, if you ask us to re-run a sequence and the results are the same as the first time, then we will have to charge you for the additional reaction. If the results are different from the first time, then we will not charge you.

 

 
 
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