DNA SEQUENZIERUNGNEXT GEN SEQUENCINGDNA/RNA OLIGOSFORSCHUNGLOGINSEQLAB

Hints and Tips

In the following you will find hints and tips on the following topics. In case this information does not answer your question(s), do not hesitate to contact us.

In the following you will find hints and tips on the following topics. In case this information does not answer your question(s), do not hesitate to contact us.

 

Premium Run vs. Economy Run Service

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The following table provides an overview of the main differences between our Premium Run Service and our (Barcode) Economy Run Service:

 

Service Features

Premium Run

Economy Run & Barcode Economy Run

Sample preparation via the customer

DNA sample and sequencing primer are to be kept separate

DNA sample and sequencing primer can be mixed or kept separate

Sample amount requirements

Is variable and depends on the number of anticipated sequencing reactions

Is fixed; a pre-defined sample amount will be required per sequencing reaction

Addition of sequencing primers at Microsynth

Standard or specific sequencing primers are added free of charge

Standard or specific sequencing primers can be added free of charge

1 tube = 1 reaction

No

Yes

Time schedule

1 day per reaction

1 day per reaction

Sequence read length per run

Up to 1‘100 bases  (~800 on average)

Up to 1‘100 bases  (~800 on average)

Editing

Your choice

Not available

Paper printout of chromatograms

Your choice

Not available

Customer support

Intensive

Basic

Troubleshooting

Yes, full support including problem-oriented solutions

No

Repetition of failed reactions

Yes

Upon request

Combination with additional useful services

Yes
Can be combined with following services:

  • Special Treatment
  • One-Drop Sequencing
  •  DNA Preparation (E. coli)
  • DNA Purification

 

Limited
Can be combined with following services:

  • Specific treatment of GC-rich samples
  • PCR Purification

 

Storage of samples

Yes
Templates: 3 months
Primers: 1 year

No

 

 

 

DNA Template Preparation

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First of all, in order to yield good sequencing results it is critical that your DNA template does not contain EDTA. EDTA inhibits the polymerase and hence precludes proper amplification of your DNA template (chain-termination method). Therefore, we highly recommend that you re-suspend your samples either in water or in 10 mM Tris buffer (pH 8.5).

 

Second, we kindly suggest you to read and consider the following points with respect to isolation/preparation of your DNA templates from E. coli cultures or after performing PCR amplification.

 

Plasmids

It is generally known that some plasmids have a low copy number in E. coli. If you have to deal with such low copy plasmids, please purify them ahead of sequencing (e.g. by means of ethanol precipitations or by applying spin columns). If you feel unsafe or if you don’t want to deal with such additional work, please let us know and instruct us to do this for you.

  

Please Note: Precipitation with ethanol after DNA isolation can improve the quality of your sequencing results!

  

PCR Products

In order to obtain reliable sequencing results, impurities such as dNTPs, PCR primers etc. must be eliminated from the PCR product before sequencing. Furthermore, it is important to quantify the purified PCR product. This is easily accomplished by performing agarose gel electrophoresis of your PCR product and comparing the band for your PCR product with standard bands of defined concentrations. We do not recommend quantification approaches based on spectrophotometric analysis techniques. For successful sequencing, PCR products must be single-banded on the agarose gel. If this is not the case, the sequencing output is likely to be useless. Therefore, purification through agarose gel is important. 


Please Note: If available, please send a picture of the gel image after the final purification step.Please check that your PCR primers meet the requirements for sequencing primers. Sometimes usage of internal sequencing primers is helpful.PCR products <100 bases are often problematic for sequencing. Instead of sequencing such PCR products directly, we recommend cloning them into a plasmid vector.

 

 

 

Shipment of Samples

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Tubes

Please use only 1.5 ml conical tubes for shipment of your samples. If possible, we recommend you to use 1.5 ml tubes with screw-caps (e.g. from Sarstedt) or with snap-caps (e.g. Safe-Lock tubes from Eppendorf), since they provide optimal protection for your samples (no accidental lid opening). Please note that due to an increasing degree of automation Seqlab-Microsynth can no longer process 0.2 ml, 0.5 ml and 2.0 ml tubes. When shipping samples to us, please insert the 1.5 samples tubes as well as your printed order form into a transparent plastic bag and drop all together into the nearest Seqlab mail box. Should there be no Seqlab mail box in your environment, please request from us prepaid and preaddressed envelopes for cost-free and convenient shipment of your samples.

 

Please note that our automated process cannot handle sample tubes which are wrapped with parafilm. Screw-cap or snap-cap tubes have a tight seal and are quite stable. Wrapping the tubes in parafilm is not necessary.

 

 

Labeling

Barcode Economy Run: Just stick the green, pre-paid barcode label on the tube.

   

Economy Run: We provide you blue barcode labels (not pre-paid!) which can be stuck on the tube for sample identification. Alternatively, tubes with handwritten labels are fine, but please write clearly and use a waterproof pen. Write on the tube, not on the lid. 

 

Premium Run: Tubes with handwritten labels are fine (please write clearly and use a waterproof pen). Write on the tube, not on the lid.

 

 

 Sequencing Primers

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With respect to the use of standard or specific sequencing primers we can offer you the following four options:

  1. You select and use one of our standard primers free of Charge
  2. You have your own sequencing primer and want to use it. All you have to do is send the primer along with your samples.
  3. You want to order a specific sequencing primer but don’t know how to design it. In this case, please provide all essential information and we will do the design and subsequent Synthesis.
  4. You want to order a specific sequencing primer. Just follow the design guidelines further below and initiate a purchase order via our webshop. Just use the “order now” option.     

 

Guidelines for Primer Design

  • Primer length should be ~20 bases (+/-2)
  • G/C content should be ~50 %
  • Avoid hairpins, palindromic sequence and dimers
  • Avoid primers with 90 % assembly with a second binding site or where the last 7 bases from the primer's 3'-end match perfectly with another site
  • Please remember that 30-60 nucleotides get lost between the 3'-end of the primer and the start of sequence. Take this into account when you choose your primer position. 

 

Primer Storage

All primers used for our Premium Run Service will be stored at our facility for 8 months. During this time these primers or aliquots of them will be sent to you only upon request. After 8 months they will be discarded without further notice.

 

 

 

 Standard Primer List

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Primer Name

Primer Sequence

3-AOX1

GCAAATGGCATTCTGACATCC

5-AOX1

GACTGGTTCCAATTGACAAGC

-96gLLL

CCTCATAGTTAGCGTAAC

AmpStart

AAAACAGGAAGGCAAAATGC

AmpStop

TCAGGCAACTATGGATGAAC

BGH-rev

TAGAAGGCACAGTCGAGG

C-CMV-24

TAGGACAAGGCTGGTGGGCAC

CEP4-for

AGCTCGTTTAGTGAACCG

CMV-for

CGCAAATGGGCGGTAGGCGTG

CMV-rev

AGTAGGAAAGTCCCGTAAGG

EBV-rev

GTGGTTTGTCCAAACTCATC

EGFP-C1-anti

GGTTCAGGGGGAGGTGTG

EGFP-C-F-31

CAAAGACCCCAACGAGAAG

EGFP-C-for

GTCCTGCTGGAGTTCGTG

EGFP-C-Rev

AGCTGCAATAAACAAGTT

EGFP-for

CAACGAGAAGCGCGATC

EGFP-N-for

CAACGGGACTTTCCAAAATG

EGFP-N-R+33

CGGACACGCTGAACTTGTG

EGFP-N-rev

GCTTGCCGTAGGTGGCATC

FastBac-for

TACTGTTTTCGTAACAGTTTTG

FastBac-rev

CATTTTATGTTTCAGGTTCAGG

GAL4-AD

TACCACTACAATGGATG

GAL4-BD

TCATCGGAAGAGAGTAG

GEX3-rev

GCTTACAGACAAGCTGTGAC

GEX3-rev-25

GAGCTGCATGTGTCAGAGG

GEX5-for

CCAGCAAGTATATAGCATGG

GL2

CTTTATGTTTTTGGCGTCTTCC

GL3

CCGGGCCTTTCTTTATGTTTTTG

IRES-for

TAGGCGTGTACGGTGGG

IRES-R-359

ACCCCAACAGCTGGCCCTCG

IRES-rev

TATAGACAAACGCACACCG

ITS1

TCCGTAGGTGAACCTGCGG

ITS4

TCCTCCGCTTATTGATATGC

LacOp-for

CGGATAACAATTTCACACAG

M13

TGTAAAACGACGGCCAG

M13-40

GTTTTCCCAGTCACGAC

M13r

CAGGAAACAGCTATGAC

malE

GGTCGTCAGACTGTCGATG

pBAD-F+50

TTATCGCAACTCTCTACTGT

pBAD-for

ATGCCATAGCATTTTTATCC

pBAD-rev

GATTTAATCTGTATCAGG

pCIneo-for

CCACTCCCAGTTCAATTACAG

pCIneo-rev

GTATCTTATCATGTCTGCTCG

pCI-rev

GCAATAGCATCACAAATTTCAC

pDONR-3

GCAATGTAACATCAGAGAT

pDONR-5

TAACGCTAGCATGGATCTC

PEN1-737R

TCCAGCTCGACCAGGAT

pENTattL1-for

TCGCGTTAACGCTAGCATGG

pENTattL2-rev

ACATCAGAGATTTTGAGACACG

pET-down

GATTATGCGGCCGTGTAC

pETG-for

CTGGCAAGCCACGTTTGG

pET-T7side

GGGAATTGTGAGCGGATAAC

pET-up

ATGCGTCCGGCGTAG

pGMP-rev

GATATAGTTCCTCCTTTCAGC

pHEN-rev

AGATCCTCTTCTGAGATG

pJET1.2-for

CGACTCACTATAGGGAG

pJET1.2-rev

ATCGATTTTCCATGGCAG

pJET1-for

CTGAACACCATATCCATCC

pJET1-rev

GCAGCTGAGAATATTGTAG

pKK223-3-for

GGCGTTTCACTTCTGAGTTC

pKK223-3-rev

CGGTTCTGGCAAATATTCTG

PolyT-A

TTTTTTTTTTTTTTTTTTTTA

pTRE-for

TAAGCAGAGCTCGTTTAGTG

pUCM13-52

GCTGCAAGGCGATTAAGTTG

pUCM13-rev-157

TGCTTCCGGCTCGTATGTTG

Puro-rev

TGTACTCGGTCATGGTAAGC

Qe30-for

CTTTCGTCTTCACCTCGAG

Qe30-rev

CCAAGCTAGCTTGGATTCTC

QE-for

GTATCACGAGGCCCTTTCG

QE-rev

GTTCTGAGGTCATTACTGG

RV3

CTAGCAAAATAGGCTGTCC

RV4

GACGATAGTCATGCCCCGC

SK

CTAGAACTAGTGGATCC

SP6

ATTTAGGTGACACTATAG

SUMO-5

CCTTAAGATTCTTGTACGACG

SV40-for

GCCCCTAACTCCGCCCATCC

SV40-pA-rev

CCTCTACAAATGTGGTATGG

T3

TTAACCCTCACTAAAGG

T7

TAATACGACTCACTATAGGG

T7probis

TCCCGCGAAATTAATACG

T7terbis

AACCCCTCAAGACCCG

T7term

TGCTAGTTATTGCTCAGCGG

TET-CMV-for

CCTCCATAGAAGACACC

U6

GGGCAGGAAGAGGGCCTAT

WHV-5R

AGCAGCGTATCCACATAGCG

XL39-rev

TAGGACAAGGCTGGTGG

 

 


Sample Amounts and Concentrations

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Premium Run Service

General Information:

Each DNA sample and each primer must have a minimum volume of 20 µl. DNA samples and primers for sequencing reactions are best dissolved in pure water. Alternatively, 10 mM Tris-HCl (pH 8) or 10 mM Tris-HCl (pH 8) with a maximum of 0.01 mM EDTA can be used for better long term DNA stability. Standard TE-buffer is not suitable, because higher EDTA concentrations inhibit sequencing polymerase activity. DNA samples will be stored for 3 months, whereas primers will be kept at Microsynth for 1 year.

 

 In order to speed up your sequencing inquiries, we offer the possibility to order sequencing primers directly in the order form (option "order now"). We are also happy to assist you in primer design. Just send us the sequence context and we design and synthesize the needed sequencing primers for you. Stored primers can be added to a custom primer list. The names of these primers are then available in a pull down menu when you order your next sequencing reaction.

 

Sample Amounts & Concentrations

DNA Template

Concentration

 

Effective Amount

(in 20 µl)

Each Additional Reaction Requires

Plasmid

100 ng/µl

2 µg

+5µl

PCR (> 5000bp)

100 ng/µl

2 µg

+5µl

PCR (< 5000bp)

40 ng/µl

0.8 µg

+5µl

PCR (< 1000bp)

20 ng/µl

0.4 µg

+5µl

PCR (< 500bp)

10 ng/µl

0.2 µg

+5µl

PCR (< 200bp)

5 ng/µl

0.1 µg

+5µl

Primer

10 pmol/µl = 10 µM

200 pmol

+5µl

The minimum amount is 20 µl. If you have sufficient primer amounts, please send us more for future sequencing reactions.

 

 

 

Economy Run and Barcode Economy Run Service

General Information:

DNA samples and sequencing primers can be sent pre-mixed (within one tube) or separate (different tubes). Each DNA sample must have a volume of 12 µl. Each sequencing primer must have a minimal volume of 20 µl whereas for each additional reaction at least 5 µl have to be added. DNA samples and primers for sequencing reactions are best dissolved in pure water. Alternatively, 10 mM Tris-HCl (pH 8) or 10 mM Tris-HCl (pH 8) with a maximum of 0.01 mM EDTA can be used for a better long term DNA stability. Standard TE-buffer is not suitable, because higher EDTA concentrations inhibit polymerase activity. Templates are stored for 1 week. Your specific sequencing primers will be kept at our sequencing lab for at least 6 months (or for 12 months in case you have added them to your "Custom Primer List").

 

Sample Amounts & Concentrations

DNA Template

Concentration

 

Effective Amount

(in 12 µl)

Pipetting Scheme (Pre-mixed)

Plasmid

40-100 ng/µl1

480-1200 ng

12 µl DNA template solution + 3 µl sequencing primer solution

PCR

18 ng per 100 bases in a volume of 12 µl

PCR (200bp)

3.0 ng/µl

36 ng

PCR (300bp)

4.5 ng/µl

54 ng

PCR (400bp)

6.0 ng/µl

72 ng

PCR (>400bp)

etc.

etc.

Primer (pre-mixed)
Primer (separate)2

2 pmol/µl
10 pmol/µl = 10 µM

30 pmol
-

 

 

 

High-Throughput and Barcode High-Throughput Sequencing Service

General Information:

Please send the DNA in 96-well plates (at least 10 µl volume). DNA samples and primers for sequencing reactions are best dissolved in pure water. Alternatively, 10 mM Tris-HCl (pH 8) or 10 mM Tris-HCl (pH 8) with a maximum of 0.01 mM EDTA can be used for a better long term DNA stability. Standard TE-buffer is not suitable, because higher EDTA concentrations inhibit sequencing polymerase activity. DNA samples will be stored for 3 months whereas primers will be kept at Microsynth for 1 year.

 

In order to speed up your sequencing inquiries, we offer the possibility to order sequencing primers directly at Microsynth. Just order the primer in the oligonucleotide order and choose the option “keep primer for sequencing” and leave a comment when you order the sequencing reactions. We are also happy to assist you in primer design. Just send us the sequence context, and we will design and synthesize the needed sequencing primers for you.

 

Sample Amounts & Concentrations

DNA Template

Amount/Concentration

Plasmid

0.8 µg

PCR

purified & unpurified PCR product: 1.5 ng/µl (per 100 bases)

Primer

20 pmol premixed (unless you use Microsynth’s standard primers or your stored specific primer). 10 µM primer aliquots for separately sent primers. The minimum amount is 200 µl. If you have sufficient primer amounts, please send us more for future sequencing reactions. New primers can also be ordered through the online order form indicating "order now" as primer source.

 

 

 

Primer Walking Sequencing Service

General Information:

DNA samples must have a minimum volume of 20 µl. Primers [10 µM] must have a minimum volume of 20 µl. DNA samples and primers for sequencing reactions are best dissolved in pure water. Alternatively, 10 mM Tris-HCl (pH 8) or 10 mM Tris-HCl (pH 8) with a maximum of 0.01 mM EDTA can be used for better long-term DNA stability. Standard TE-buffer is not suitable, because higher EDTA concentrations inhibit sequencing polymerase activity. DNA samples will be stored for 3 months whereas primers will be kept at Microsynth for 1 year.

 

Alternatively, you can send your E. coli strain. DNA is isolated free of charge for all Primer Walking Services on plasmids. The design and synthesis of walking primers is included in the service.

 

Sample Amounts & Concentrations

DNA Template

Amount/Concentration

Plasmid for Single-Stranded Sequencing

2 µg of plasmid-DNA for a 1 kb insert and another 1 µg for each additional kb

Plasmid for Double-Stranded Sequencing

4 µg of plasmid-DNA for a 1 kb insert and another 2 µg for each additional kb

 

 

Chromatogram Evaluation

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It is always advisable to check the quality of the chromatogram by eye. For this purpose we recommend you to download one of the Software tools for visualization in our Software and Links area (Note: this link will transfer you to the Microsynth Website).

 




1 In order to yield good sequencing results, please adjust your DNA plasmid solution within the requested concentration range. Please consider that the optimal DNA concentration is 80 ng/µl.


2 Direct primer synthesis for sequencing possible.

Seqlab - Sequence Laboratories Göttingen GmbH
Hannah-Vogt-Str. 1
37085 Göttingen

Postfach 3343
37023 Göttingen

Telefon 0551-370 00 10
Telefax 0551-370 00 12

E-Mail info@seqlab.de

Dear Customer,

we are pleased to announce our new Trouble Shooting Guide. Inside you will find detailed descriptions of common problems leading to inferior sequencing results.

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