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Macrogen has served over 10 years in sequencing
field using the cutting edge technology and
delivering fast and reliable results. We
use state-of-the-art high throughput Applied
Biosystems 3730XL sequencers.
We sequence all kinds of cloned DNAs (plasmids,
cosmid, phages, BACs) as well as PCR-products.
Macrogen provides a high quality DNA Sequencing
service for academics, government, research
institutions and private companies at Very
Competitive Price.
Please contact us for any inquiry.
For more detailed explanations and recommendations
about the preparation and submission of samples,
please refer to the following link:
For information regarding to the shipping
specifications, please refer to the following
link
 Download
'Shipping Information for Value Service to Macrogen Inc. (Korea)'
Download
'Shipping Information for European Express Service to Macrogen Europe
(The Netherlands)'
http://dna.macrogen.com/eng/help/orderg.jsp
To order the sequencing service, please
access to our online ordering system and create an account;
http://dna.macrogen.com/eng
For payment information, please refer
to the following link:
Download
'Payment Information'
http://dna.macrogen.com/eng/help/howtopay.jsp
Contact
Sequencing Inquiry: info@macrogen.com
Payment Inquiry: payment@macrogen.com
Genome and Project sequencing inquiry: ngs@macrogen.com
Technical Support
S.H. Cha, Ph.D : chash@macrogen.co.kr
Shipping Address
(for Value Service to Macrogen Inc.)
Macrogen Inc.
908 World Meridian Venture Center
#60-24,
Gasan-dong,
Geumchun-gu
Seoul 153-781, Korea
Tel : +82-2-2113-7790
Fax : +82-2-2113-7919
E-mail : info@macrogen.com
Shipping Address (For
European Express Service to Macrogen Europe)
[Macorgen Europe (The Netherlands) ]
IWO, Kamer IA3-195
Meibergdreef 39
1105 AZ Amsterdam Zuid-oost
Netherlands
Tel : +31-(0)20-566-5472
Mobile : +31-(0)6-8148-0760
E-mail : europe@macrogen.com
Template
Type/Format |
Sample Requirements |
Remarks |
| Plasmid |
- 100 ng/µl
- Minimum volume of 20µl |
For re-sequencing, at lease
5 µl is required |
| 16S |
- Agar Plate/Glycerol Stock
- gDNA: 30-50ng
- Minimum volume of 20µl |
N/A |
PCR Product
(Purified) |
- 50ng/µl
- Minimum volume of 20µl |
For re-sequencing, at lease
5 µl is required |
PCR Product
(Unpurified) |
- 100 ng/µl
- Minimum volume of 30µl |
N/A |
| Difficult Sequence |
- 100 ng/µl
- Minimum volume of 40µl |
N/A |
| Primer Walking |
- 8 µg(for an insert
size of up to 4kb)
|
Single Strand Sequencing:
1µg/1 kb
insert.
If insert size is longer than 4kb,
clone is required in an agar stab
culture status. |
|
| a)
Sample for individual tube
: |
- General glycerol
stock/ Agar-stab/Agar-plate culture
at room temperature.
- 1.5µl microcentrifuge tube is recommended in a dried (lyophilized)
form or solution
..(Nuclease-free
TE or distilled water) at room
temperature..
- Free re-sequencing is included |
| b)
96 Well Plate : |
- 8 strip cap
is recommended in a dried (lyophilized)
form or solution (Nuclease-free
TE or distilled water)
.. at
room temperature.
- Re-sequencing is
additionally charged.
** In case of Multi primers for one plate **
Only two custom primers (or two universal primers) in tubes could be acceptable
for applying this Plate sequencing.
* Note:
- For three or more primers, another paired primer plate with your sample….reactions.
- The well to well variation of the concentration of your samples is very important
to get
better results, please keep it as constant as possible.
- Please prepare samples to avoid
any well-to-well concentration
difference or size differencefor
quality results.
|
| c)
Primer : |
- Primer : 10
pmol/uL in 20µl based on
5 samples
- Primer Tm is suitable for service : 55°C ~ 60°C
- Commonly used Universal primers are available for free of charge
(ex. M13F, T7, T3)
- Design and synthesize primers if you request. (US $0.60/base) |
It is possible to send either a stab or glycerol
culture with the selected E.coli clone,
the purified plasmid DNA or your unpurified
or purified PCR product.
Templates and primers must be provided in
DI water or 10mM Tris buffer, not in TE |
| d) To apply Plate sequencing : |
- 1 primer/1plate service :
- Multi primers/ 1plate service :
Only
two custom primers (or two universal
primers) in tubes could be acceptable
for applying this Plate
sequencing.
Note : For three or more primers, another paired primer plate with your sample
plate should be provided. If not, your order will be regarded as Normal sequencing
reactions. |
 |
 DNA / RNA Quality
a) DNA |
All DNA sample(s) requirement...
-double-strand, non degraded, and containing
no particulate matter
-purified by column or gel purification protocols
-A260/280 ratio of 1.8 or above |
| b) RNA |
All RNA sample(s) requirement...
-2100 Bioanalyzer RNA integrity Number (RIN) > 8
-28S rRNA : 18S rRNA = 2:1 ratio
|
DNA / RNA / Tissue Quantity
a) 454 GS FLX (in EB Buffer) |
| HMW(>1.5kb) |
LMW(70~500bp) |
Paired End |
| 5~10µg |
5µg |
5~10µg |
* All samples except for LMW(=50ng/µl) should be provided at a concentration
of =100ng/µl
In case of cDNA synthesis, tissue(>10g) or total RNA (>1mg) would be required. |
| b) Illumina Genome Analyzer
( in EB buffer) |
| Sample Type |
Total Quantity |
| gDNA (for single/paired-end run) |
5µg (recommended) |
| ChIPed DNA |
10ng |
| mRNA sequencing-total RNA |
1~10µg |
| mRNA sequencing-mRNA |
100ng |
| small RNA sequencing-total RNA |
1~10µg |
| small RNA sequencing-small RNA |
entire fraction of small RNA
purified from
1~10µg of total
RNA |
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1) Template preparation
The success of automated sequencing critically
depends on having high purity template in
the correct concentration. In case of samples in plates, constant
concentration among wells is also important for the success of sequencings.
i) Plasmid DNA
Preparation
There are many commercial kits
available. Please submit DNA
in deionized water. Do not use
TE to dilute or resuspend the
DNA because EDTA inhibits the
cycle of sequencing reaction.
We recommend using Qiagen miniprep/midiprep
since both methods yield consistent
purity of plasmid DNA for sequencing.
Please provide DNA in the concentration
range of 100ng/µl and in the amount of at least 2 µg.
Extra amount of DNA ensures that
we have enough sample for a re-sequencing
in case the first reaction fails.
If samples' concentrations do
not fall within this range or if you fail to provide us enough
template to do the reaction, the experiment might be delayed. |
ii) PCR fragments
Preparation
The DNA is free of contaminants,
unused primers or dNTPs. PCR
templates that do not undergo
any kind of post PCR clean up are not suitable for sequencing and
will yield
unusable sequence data. It is
highly recommended that your
PCR template is first observed on a gel to confirm that there is
a specific product
with the correct size. The Qiagen
Gel extraction kit or PCR cleanup
kit can be used to remove all of the unwanted elements from your
template. |
2) Host strains
The host strain can have an impact on the
quality of the template DNA prepared evenusing
the best methods.
DH5-α strains consistently produce
good results. HB101, XL-1 Blue, JM109 and
MV1190
are usually fine but JM101 is often poor.
The growth media you use can also affect
the outcome yields, while LB is usually fine.
3) Quantitation
Sequencers are able to handle a wide range
of DNA concentrations however with very low
amounts of DNA the data quality will be significantly
affected.
Using UV absorbance to quantitate dilute
DNA solutions tends to give widely inaccurate
results.
A good way to quantitate
DNA is to run an aliquot on a minigel and
compare the intensity to the a control of
a known concentration. There
are also concentration ladders that are commercially
available. For each reaction, please provide
10 ng/100 bases, and at least 20 ng/µl
solution in deionized water. Please provide
at least 10 µl for any possible re-sequencing.
Measurement with electrophoresis is recommended rather than spectrophotometer-based measurements.
4) Primers preparation
Primer
Considerations
Primers should be provided in DI water at the required concentration
(see table above).
- High Purity
- Appropriate concentration
- No secondary priming sites
- No mismatches
- A length of 18-25 bases.
- GC% content between 40% and 60%.
- A Tm (melting temperature) between 55°C and 60°C
- No significant hairpins (>3bp)
- Free of salts, EDTA, or other contaminants
Please supply primers at concentration of (10 pmole/µl =60
ng/µl) in deionized water at volume of greater than 20 µl.
Primers supplied by customers should be desalted or purified. Crude
primers generally do not work well for sequencing. We have the following
primers available at no extra charge.
| Primer
Name |
Sequence |
Base |
| T7 |
AATACGACTCACTATAG |
17 |
| T7terminator |
GCTAGTTATTGCTCAGCGG |
19 |
| T7promoter |
TAATACGACTCACTATAGGG |
20 |
| T3 |
ATTAACCCTCACTAAAG |
17 |
| SP6 |
ATTTAGGTGACACTATAG |
18 |
| M13F-pUC(-40) |
GTTTTCCCAGTCACGAC |
17 |
| M13R-pUC(-40) |
CAGGAAACAGCTATGAC |
17 |
| M13F(-20) |
GTAAAACGACGGCCAGT |
17 |
| M13R(-20) |
GCGGATAACAATTTCACACAGG |
22 |
| pGEX5 |
GGCAAGCCACGTTTGGTG |
18 |
| pGEX3 |
GGAGCTGCATGTGTCAGAGG |
20 |
| 27F |
AGAGTTTGATCMTGGCTCAG |
20 |
| 1492R |
TACGGYTACCTTGTTACGACTT |
22 |
| 518F |
CCAGCAGCCGCGGTAATACG |
20 |
| 800R |
TACCAGGGTATCTAATCC |
18 |
| BGH-R |
TAGAAGGCACAGTCGAGG |
18 |
| CMV-F |
CGCAAATGGGCGGTAGGCGTG |
21 |
| RVprimer3 |
CTAGCAAAATAGGCTGTCCC |
20 |
| RVprimer4 |
GACGATAGTCATGCCCCGCG |
20 |
| GLprimer1 |
TGTATCTTATGGTACTGTAACTG |
23 |
| GLprimer2 |
CTTTATGTTTTTGGCGTCTTCCA |
23 |
| pQE-F |
CCCGAAAAGTGCCACCTG |
18 |
| pQE-R |
GTTCTGAGGTCATTACTGG |
19 |
| Gal4AD |
TACCACTACAATGGATG |
17 |
| pBAD-F |
ATGCCATAGCATTTTTATCCA |
21 |
| pBAD-R |
GATTTAATCTGTATCAGG |
18 |
| EGFP-CF |
AGCACCCAGTCCGCCCTGAGC |
21 |
| EGFP-CR |
CGTCCATGCCGAGAGTG |
17 |
| EGFP-NR |
CGTCGCCGTCCAGCTC |
16 |
| Bluescript SK |
CGCTCTAGAACTAGTGGATC |
20 |
| EBV-RP |
GTGGTTTGTCCAAACTCATC |
20 |
| KAN2-FP |
ACCTACAACAAAGCTCTCATCAACC |
25 |
| KAN2-RP |
GCAATGTAACATCAGAGATTTTGAG |
25 |
| M13-FP |
TGTAAAACGACGGCCAGT |
18 |
| pBacPAC-RP |
GTCTGTAAATCAACAACGC |
19 |
| pBAD-FP |
ATGCCATAGCATTTTTATCC |
20 |
| pDONOR-FP |
TAACGCTAGCATGGATCTC |
19 |
| pEGFP_N |
CCGTCCAGCTCGACCAG |
17 |
| pEGFP-FP |
TTTAGTGAACCGTCAGATC |
19 |
| pEGFP-RP |
AACAGCTCCTCGCCCTTG |
18 |
| pESP-RP |
TCCAAAAGAAGTCGAGTGG |
19 |
| pET-24a |
GGGTTATGCTAGTTATTGCTCAG |
23 |
| pET-RP |
CTAGTTATTGCTCAGCGG |
18 |
| pMalE |
TCAGACTGTCGATGAAGC |
18 |
| pREP-fwd_primer |
GCTCGATACAATAAACGCC |
19 |
| 35S-A |
AAGGGTCTTGCGAAGGATAG |
20 |
| 35S-B |
AGTGGAAAAGGAAGGTGGCT |
20 |
| AD Reverse |
AGATGGTGCACGATGCACAG |
20 |
| CYC1 Reverse |
GCGTGAATGTAAGCGTGAC |
19 |
| DsRed1-C |
AGCTGGACATCACCTCCCACAACG |
24 |
| DsRed1-N |
GTACTGGAACTGGGGGGACAG |
21 |
| EGFP-C |
CATGGTCCTGCTGGAGTTCGTG |
22 |
| EGFP-N |
CGTCGCCGTCCAGCTCGACCAG |
22 |
| GAL1 Forward |
AATATACCTCTATACTTTAACGTC |
24 |
| U-19mer Primer |
GTTTTCCCAGTCACGACGT |
19 |
| T7 EEV |
ATGTCGTAATAACCCCGCCCCG |
22 |
| Bluescript KS |
TCGAGGTCGACGGTATC |
17 |
| pFastBac Forward |
GGATTATTCATACCGTCCCA |
20 |
| pFastBac Reverse |
CAAATGTGGTATGGCTGATT |
20 |
| AOX1 Forward |
GACTGGTTCCAATTGACAAGC |
21 |
| AOX1 Reverse |
GCAAATGGCATTCTGACATCC |
21 |
| a-Factor |
TACTATTGCCAGCATTGCTGC |
21 |
| STag 18mer Primer |
GAACGCCAGCACATGGAC |
18 |
| MT Forward |
CATCTCAGTGCAACTAAA |
18 |
| QE Promoter |
CCGAAAAGTGCCACCTG |
17 |
| pRH Forward |
CTGTCTCTATACTCCCCTATAG |
22 |
| pRH Reverse |
CAAAATTCAATAGTTACTATCGC |
23 |
| SV40-pArev |
CCTCTACAAATGTGGTATGG |
20 |
| SV40-Promoter |
GCCCCTAACTCCGCCCATCC |
20 |
| pTrcHis Forward |
GAGGTATATATTAATGTATCG |
21 |
| ITS1 |
TCCGTAGGTGAACCTGCGG |
19 |
| ITS2 |
GCTGCGTTCTTCATCGATGC |
20 |
| ITS3 |
GCATCGATGAAGAACGCAGC |
20 |
| ITS4 |
TCCTCCGCTTATTGATATGC |
20 |
| pJET1.2R |
AAGAACATCGATTTTCCATGGCAG |
24 |
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