• How many bases can I expect to obtain?
• What is the turn-around time?
• How is the data distributed?
• How to sequence through difficult structure regions?
• Which universal primer do you provide?
• How to print out electropherograph from my computer?
• Does bacterial host strains have impact for DNA sequencing quality?
• Which commercial methods are preferred for DNA preparation?
• What precautions shall I consider for preparing DNA for sequencing purpose?
• How to evaluate the sequencing result?
• How to start trouble shooting for failed sequencing?

How many bases can I expect to obtain?
Usually 850 bases can be obtained. Good quality template may yield more. Lower quality template can significantly reduce the reading length.

What is the turn-around time?
Submit samples before 2:30pm helps you to get results next morning. Results otherwise will be ready the morning 2 days after your submission (submit on Monday after 2:30pm, ready on Wednesday morning).

How is the data distributed?
On the "Sequencing Order Form", you have three choices:
Email text file: we attach GATC text file. (link)
Print electropherogram: we print the electropherogram file, and you pick up the printout.
Attach electropherogram: we attach electropherogram file via email. (link) You need to down load the free software. The web address are: 4Peaks for MAC: http://www.mekentosj.com/4peaks, or
Sequence Scanner for PC: https://products.appliedbiosystems.com/ab/en/US/adirect/
ab?cmd=catNavigate2&catID=600583

How to sequence through difficult structure regions?
Difficult structure region includes repetitive DNA, homopolymer region (poly A, T), GC rich template (>70%), and secondary structure (inverted repeats and palindromes). They cause drops in signal intensity and noisy data immediately after the region. Sequencing the complimentary strand helps to elucidate the ambiguous data after a difficult sequencing region. Order us to use dGTP kit might improve the difficult region sequencing, although overall sequencing quality is compromised.

Which universal primer do you provide?
M13F: GTA AAA CGA CGG CCA GT (17mer, 52C)
M13R: GGA AAC AGC TAT GAC CAT G (19mer, 56C)
T7: TAA TAC GAC TCA CTA TAG GG (20mer, 56C)
T3: ATT AAC CCT CAC TAA TGG GA (20mer, 56C)
SP6: CAT ACG ATT TAG GTG ACA CTA TAG (24mer, 66C)
T7T: GCT AGT TAT TGC TCA GCG G (19mer, 58C)

How to print out electropherograph from my computer?
Panels per page: 6
Points per panel: 1500
PostScrip Printer

Does bacterial host strains have impact for DNA sequencing quality?
DH5 alpha host strains consistently produce good results.
HB101, MV1190, JM109 and XL1 Blue host strains show some variability in result quality. XL1 Blue grows slower than most strains and can lead to decreased DNA yields and it does not respond to TB as do other strains (only showing a 2-3 fold increase in cell number per ml). JM101 is not recommended.

Which commercial methods are preferred for DNA Preparation?
Recommended:
Qiagen Qiawell, Tips, Beads
PE ABD Columns
Edge Technology columns
Nucleobond AX
Promega Wizard Plus

Not Recommended:
Bio101 GeneKleen
Promega Wizard
BioRad Prep A Gene

Template preparation or purification procedures involving the use of phenol or chloroform should be avoided if possible. If use of phenol or chloroform can not be avoided an additional ethanol precipitation is recommended.

What precautions shall I consider for preparing DNA for sequencing purpose?
Do not use TE to dissolve DNA: EDTA inhibits sequencing reaction. Use ddH2O instead. (Link)

Template purity is more important than template quantity. The importance of getting rid of salt, detergent, ethanol, phenol, chloroform, PCR primer, dNTP from DNA can not be over emphasized. If the concentration of DNA is low, using 1/2 of recommended amount DNA is preferred than possibly carrying a lot of contaminants into sequencing reaction.

The directions for cell growth shall be followed (cautious use of Terrificbroth). Example: Overloading the Qiagen column yields poor quality DNA.

RNA competes with plasmid DNA binding to the Qiagen resin. It also affects the quantification of the DNA if this is done spectrophotometrically. The presence of RNA contaminants can be determined by analysis of the template preparation on agarose gels.

When estimating the amount of DNA on an agarose gel, be sure to load a low amount of DNA (i.e. 30ng-50ng per lane) with a marker of known concentration (not degraded itself). Low film exposures (∼1-2 sec) and an OD (260) is also advised. Overloaded gels and longer film exposures will likely give poor estimations of amount.

Spectrophotometer: the A (260/280) ratio should be 1.7-1.9. Smaller ratios usually indicate contamination by protein or organic chemicals. RNA contamination will affect DNA quantitation greatly. Absorbance measurements of highly concentrated (O.D. > 1.0), very dilute (O.D. > 0.05) DNA sample, and PCR products can be inaccurate.

One A (260) unit of single stranded DNA contains 33 ng/ul.
One A (260) unit of double stranded DNA contains 50 ng/ul.

Neither gel nor OD show the presence of salts, residual ethanol or EDTA contamination. Salts can be removed with spin columns such as Centri-sep (Princeton Separations) before sequencing. The isopropanol-precipitated DNA shall be washed with 70% ethanol to remove excess salt. Wash the DNA pellet at least once as directed with 70% ethanol.

The template DNA shall be dried completely before final resuspension in ddH2O. To remove residual ethanol, dry the DNA for 5 minutes in a properly operating speedvac. If air-drying, make sure that the DNA is dry (no fluid in the tube, the DNA pellet does not look wet). When air drying, a brief 15 min incubation of the open tube at 65°C is often sufficient to completely dry.

For more helpful hints check out The QIAGEN Guide to Template Purification and DNA Sequencing. http://www1.qiagen.com/literature/handbooks/INT/cleanlit.aspx.

How to evaluate the sequencing result?
First of all, we recommend you to download the free software to enable you view and edit the electropherograph file on your own computer and order us to send you the results in the form of electropherograph when fill in the "sequencing order form".

Once the electropherograph file is opened, peak height uniformity, peak separation, average signal intensity should be looked at.

Peak height uniformity can be generally in three patterns:

• Good result shows peak height evenly distributed from the beginning to the end.
• Peak height is gradually reduced as the length of the sequencing increases, usually indicating that extension of sequencing products is inhibited due to contaminants (salts, ethanol, phenol, chloroform, detergent) or that the reagents for sequencing is exhausted due to huge amounts of the DNA templates/primer submitted.
• Peak height suddenly drops at certain point, often seen at difficult structure region.

Good peak separation shows that each peak is well separated from each other and rises up from separated base. Compressed peak separation shows peaks partially overlapping with each other as shoulder-by-shoulder pattern. Severe contamination of salt, cellular component, detergents and ethanol compresses sequencing products migrating orderly through hair-thin capillary.

Check Average signal intensity (ASI) by clicking "A" icon or "I" icon at the bottom of electropherogram file. ASI around 600 is a decent number. In case ASI is <40, software would call it as "failed sequencing". Most times, we check these files. If we find that "failed sequencing" actually contains somewhat useful information, we would call it "almost failed". You shall further check the purity and quantity of your DNA and other possible factors contributing to this almost failed sequencing, rather than hoping we would be able to produce the same OK quality results in the future. ASI >1000 is not necessarily, and you can use less DNA in future.

We have control sample sequenced every time we start a sequencing reaction along with your samples, it usually has ASI around 1000.

You can use "tab" key to track ambiguous nucleotide reading where N is presented and edit it.

Sometimes, artificial T or G peak around 30 or/and 70nt region is seen due to failure in completely filtrating out the unincorporated Big-Dye Terminator. The peak is overwhelmingly high, and does not show as an individual peak but "mountain".

Text file of the result can be obtained by clicking "GATC" icon at the bottom of electropherogram file.

How shall I start trouble shooting for failed sequencing?
Refer to "What precautions shall I consider for preparing DNA for Sequencing Purpose". If you believe that your DNA quality/quantity are good, read "Possible Reasons for Failed Sequencing' might help you to do the comprehensive trouble shooting.