Frequently Asked Questions About DNA Sequencing
- Why didn't my sequencing work?
- What is Laragen's policy for repeat sequences?
- How can I retrieve my sequences?
- How many base pairs can I get from a single run?
- How close can I read from the primer?
- How can I send my samples to Laragen?
- What is the turnaround time?
- How can I get BigDye reaction mix from you?
- What about my sequencing primers?
- How can I purify my cycle sequencing products?
- How long do you keep my DNA samples and primers?
- What cycle sequencing parameters should I use for the GeneAmp PCR System?
- How can I prepare cycle sequencing reactions?
- How can I view AB trace files?
- Why is the DNA concentration you measured lower than measured by nanodrop?
Why didn't my sequencing work?
- Poor quality of template DNA: Contaminants in the DNA prep will interfere with the reaction and cause failures, noise and miscalls. Contaminants are ethanol, protein, salt, PEG and genomic DNA. We recommend Qiaprep Spin Column miniprep kit to prepare plasmid DNA. Do not overgrow the cultures and do not overload the spin column.
- Not enough DNA: Insufficient DNA will lead to insufficient signals or failed reactions. On the other side, too much DNA will also interfere with the reaction.
- Primer concentration: 10 pmole of primer is required per sequencing reaction. Make sure your primer concentrationsare correct. Too much or too little primer may result in sequencing failures. Based on our experience, you should test your sequencing primer as a PCR primer first. If the primer works as a PCR primer, it usually works fine as a sequencing primer.
- Difficult Templates: Some templates are difficult to sequence. For example, it is difficult to sequence a GC righ DNA template with regular BigDye premix. If that is the case, we will use Laragen's proprietary chemistry for difficult templates to get around it.
What is Laragen's policy for repeat sequences?
- We will provide one free repeat for failed reactions or for sequences you are not satisfied with.
How can I retrieve my sequences?
- After your sequences are ready, you will receive a notification email. You can login into your account at sequencing.laragen.com to download your sequences.
How many base pairs can I get per read?
- It depends on the quality of the DNA templates and the sequences. You can get > 900 bp per read on average. With the aid of LongTrace software, you can get up to 1100 bp per read if the quality of the sequences are good.
How close can I read from the primer?
- It depends on the quality of the DNA templates. You can get reliable sequences starting from 17 - 25 bp away from the primer.
How can I ship my samples to Laragen?
- You can ship your DNA sample either in water or EB buffer (No EDTA) by US mail or FedEx (preferred) at room temperature to:
Laragen, Inc.,
10601 Virginia Ave,
Culver City, CA 90232.- We also provide free pick-up in the Los Angeles Metro area.
- For samples in the tubes, please wrap your 1.5 ml eppendorf tubes or PCR 8 strip tubes with paramfilm and place the tubes in 50 ml conical tubes or a box to protect samples. Please arrange samples by the columns, not by the rows. You can use upload and import file functions in our DNALIMS system to upload 96-well plate format. No spaces or illegal characters are allowed in the DNALIMS. Allowable characters include: a - z, A - Z, 0 - 9, and hyphens. You can use this template for file upload.
- Please seal the 96-well plate with caps, foil tape or heat seal tape to avoid possible cross contamination and sample evaporation. Please label on the side of plate. Do not label on the top (seal or caps) of the plates.
What is the turnaround time?
Services Samples Turnaround Time
GenotypingCan I get BigDye reaction mix from you?
- NO!
What about my sequencing primers?
- We provide 14 universal sequencing primers free of charge. For customized primers, you can order them from any major oligo manufacturers. Generally speaking, the oligonucleotides used as sequencing primers do not need extra purification. The working concentration for sequencing primers should be at 10 pmol/ul or 10 uM.
Standard Primer Sequences M13 Forward GTA AAA CGA CGG CCA GT M13 Reverse CAG GAA ACA GCT ATG AC T7 Promoter TAA TAC GAC TCA CTA TAG GG T3 ATT AAC CCT CAC TAA AG SP6 ATT TAG GTG ACA CTA TAG T7 Terminator CTA GTT ATT GCT CAG CGG TG pGEX 5' GGG CTG GCA AGC CAC GTT TGG TG pGEX 3' CCG GGA GCT GCA TGT GTC AGA GG RV3 CTA GCA AAA TAG GCT GTC CCC BGH Reverse TAG AAG GCA CAG TCG AGG C pBADfor ATG CCA TAG CTT TTT ATC C pBADrev GAT TTA ATC TGT ATC AGG pFastBacFwd TAT TCC GGA TTA TTC ATA CCG TC pFastBacRev GTA TGG CTG ATT ATG ATC CTC CMVfor CGC AAA TGG GCG GTA GGC GTG CMVRev AGT AGG AAA GTC CCG TAA GG How can I purify my cycle sequencing products?
- We use our own proprietary BigDye removal technique to remove unused BigDye from sequencing reactions.
How long do you keep my DNA samples and primers?
- Unless you specifically request, we normally keep our clients' DNA samples and primers for three weeks. The DNA samples and primers will be trashed after three weeks.
What are the cycle sequencing parameters I should use for the GeneAmp PCR System?
- To set up cycle sequencing reaction with the GeneAmp PCR System (2400, 9600, 9700) with BigDye 3.1, use the follow parameters (Adapted from PE Biosystems):
How can I prepare cycle sequencing reactions?
Free Software to view AB trace filesTo prepare the reaction mixtures, use following amount of DNA depending on templates (Modified from PE Biosystems):
Step Reaction 1 For each reaction, add the following reagents to a separate tube: Reagents Amount Template single strand DNA 1 ul of DNA at 50 - 100 ng/ul double strand DNA 1 ul of DNA at 100 - 200 ng/ul PCR product DNA 0.5 ul of DNA at 10 ng/ul (5 ng per 100 bp) Primer 1 ul of Primer at 10 uM (10 pmol) BigDye 3.1 1.0 ul 5x Sequencing Dilution Buffer 1.2 ul sequencing grade water q.s Total Volume 10 ul 2 Mix well and spin briefly
- FinchTV. FinchTV is easy to use and is able to view raw data.
- Sequence Scanner. Sequence Scanner is a powerful software to view AB trace file. Sequence Scanner is able to provide a variety of QC data and is able to view multiple trace files.
Why is the DNA concentration you measured lower than measured by nanodrop?
- We use a fluorometer coupled with Hoechst 33342 dye to measure DNA concentration. Because Hoechst 33342 only binds to double strand DNA, not protein, RNA or other contaminants, the DNA concentration measured with this technique is more accurately measured. In comparison, nanodrop is based on absorption peak at 260/280 nm wavelength. Many contaminants in the DNA solution could affect the OD reading. We found if the DNA concentration measured by fluorometer is significantly lower than the client's concentration measured by spectrophotometer (such as nanodrop) the sequencing reactions tends to fail due to the contaminants present in the DNA samples.