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Why my sequencing didn't 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.
- Not enough DNA: Insufficient DNA will lead to insufficient signals or failed reactions. On the flip side, too much DNA will also interfere with the reaction.
- Primer concentration: 5 pmole of primer is required per sequencing reaction. Make sure your primer concentrations are 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 dGTP BigDye chemistry to get around it.
What is your policy for repeat sequences?
- We will provide one free repeat for failed reactions.
How can I retrieve my sequences?
- You can download your sequences directly from our DNALIMS server. Please refer our download page for detail information..
How many base pairs can I get per read?
- Depends on quality of DNA templates, we can get > 900 bp per read. With aid of LongTrace software, you can get upto 1100 bp per read.
How close can I read from the primer?
- Depends on quality of DNA templates, we can get reliable sequence starting from 17 - 25 bp away from the 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. Please place all ependorf tubes in 50 ml conical tubes. Our mailing address is Laragen, Inc., 10755 Venice Blvd, Los Angeles, CA 90034.
What is the turnaround time?
Type of Sequencing Turnaround Time Ready-to-run Next day Sequencing from purified templates Next day Full services sequencing 24 to 48 hours Can I get BigDye reaction mix from you?
- BigDye reaction mix is available to our UCLA customers only. Just fill out the Sequencing Request Form and submit it. We will deliver the mix to you next day.
What about my sequencing primers?
- We provide 10 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
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 How can I purify my cycle sequencing products?
- We use Applied Biosystems' Xterminator to clean up BigDye 3.1 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 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):
Step Sequencing reaction-Plasmid/PCR prodcuts Sequencing reaction BAC/Cosmic Clones 1
10 ul Reaction Volume. 20 ul Reaction Volume. 2
Heat the tubes at 96 oC for 1 minute Heat the tubes at 96 oC for 5 minutes Repeat the following for 25 cycles Repeat the following for 50 cycles * Rapid thermal ramp to 96 oC * Rapid thermal ramp to 96oC * 96 oC for 10 sec * 96 oC for 30 sec * Rapid thermal ramp to 50 oC * Rapid thermal ramp to 50 to 55 oC * 50 oC for 5 sec * 50 to 55 oC for 10 sec * Rapid thermal ramp to 60 oC * Rapid thermal ramp to 60 oC * 60 oC for 4 min * 60 oC for 4 min 3
Rapid thermal ramp to 4oC and hold until ready to purify. 4
Spin down the contents of the tubes in a microcentrifuge. 5
Proceed purification step (Laragen provides free Bigdye Purification) Note: Rapid thermal ramp is 1oC per sec.
How can I prepare cycle sequencing reactions?
To 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 Quantity Template single strand DNA 50 - 100 ng double strand DNA 100 - 200 ng PCR product DNA 30 - 90 ng (10 ng per 100 bp) Primer 5 pmol BigDye 3.1 1.0 ul 5x Sequencing Dilution Buffer 1.5 ul sequencing grade water q.s Total Volume 10 ul 2 Mix well and spin briefly
- Free Software to view AB trace files
- FinchTV. FinchTV is easy to use and is able to view raw data.
- Sequence Scanner. Sequence Scanner is a very 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.
- Some old version software related to DNA sequence analysis
- For MacIntosh computer users, you can use PE Applied Biosystems' Editview. Editview is a free software that allows you to view, edit and print sample files (chromatograms). For more information about Editview, please refer to PE Applied Biosystems' Editview homepage . You can download Editview
from this site.
- For Windows based users, you can use Conor McCarthy's Chromas. Chromas is a free software (up to version 1.45) which allows you to view, edit and print sample files (chromatograms). However, newer versions of Chromas are available as shareware. For more information about Chromas, please refer to Chromas's homepage. You can download Chromas 1.45 (16 bit) or Chromas 1.45 (32 bit) from this site.
- To convert electropherogram files from PC/UNIX format back to MacIntosh format, you need to download the ABI File Type Converter.
- Stuffit Expander Version 7.0 for Windows.
- PE Applied Biosystems DNA Sequencing Related Documents
- DNA sequencing manual PDF file
- DNA sequencing chemistry guide PDF file
- Qiagen DNA Sequencing Related Documents
- The QIAGEN Guide to Template Purification and DNA Sequencing (2nd Edition) PDF file
- The art of plasmid purification PDF file
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