How to Prepare Samples for Macrogen Sanger Sequencing

Macrogen provides Sanger Sequencing for a full range of cloned DNA samples and PCR products.
Simply place your order online and insert your samples together with a print out of your Online Order
Form in a Syntezza supplied Ziploc bag. A Syntezza representative will pick up your samples and you
will receive an email containing a confidential link to your sequencing results.

Templates and primers should be prepared in accordance with the following chart:

Preparing samples and primers

Primer Volume
Sample Sample
Template or Service + Primer
Concentration Volume*
Concentration

Plasmid 3-4 kb 100~150ng /µl Minimum volume 20µl 5µl + 5pmo/µl

Plasmid 4–10 kb 150ng /µl Minimum volume 20µl 5µl + 5pmo/µl

Plasmid 10-15 kb
500ng /µl Minimum volume 20µl 5µl + 5pmo/µl
Note: 15kb is the maximum plasmid

PCR product < 300bp

10-15ng /µl Minimum volume 20µl 5µl + 5pmo/µl
Purified

PCR product 300-700bp

25-50ng /µl Minimum volume 20µl 5µl + 5pmo/µl
Purified

PCR product >700bp

> 50ng /µl Minimum volume 20µl 5µl + 5pmo/µl
Purified

PCR product < 300bp

20-25ng /µl Minimum volume 30µl 5µl + 5pmo/µl
Unpurified

PCR product 300-700bp

50ng /µl Minimum volume 30µl 5µl + 5pmo/µl
Unpurified

PCR product >700bp

> 50ng /µl Minimum volume 30µl 5µl + 5pmo/µl
Unpurified

BAC > 500ng /µl Minimum volume 20µl 5µl + 5pmo/µl

gDNA Full Sequencing 16S/18S/26S

rRNA or ITS region (supplied in Agar gDNA: 30-50ng Minimum volume 50µl
Plate or Glycerol Stock)

Difficult Sequencing 100 ng /µl Minimum volume 40µl

8/µg
Single Strand Primer Walking < 4kb Minimum volume 50µl
1µg /1kb insert

Primer Walking > 4kb Inquire for details. Requires clone in agar stab culture.

* Macrogen uses a minimum of 5µl for the first reaction and uses the remaining sample for
additional reactions, failed reaction or re-sequencing.

1
Templates consisting of purified plasmid DNA or PCR product, as well as primers may be
submitted in individual tubes or in a 96-well plate, in solution or dried (oven dried or
lyophilized). Samples submitted in solution may be sent in Nuclease-Free distilled water,
10mM Tris buffer or TE. Templates and primers may be submitted at room temperature.
If you wish to submit bacteria containing plasmid in general glycerol stock, agar-stab or agar-
plate culture, please contact us for additional instruction at 02-586-7138 or by email
([email protected]).

Template Preparation
The success of automated sequencing critically depends on having high purity template in
the correct concentration.
Plasmid DNA
There are many commercial kits available. We recommend using Qiagen miniprep or
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 2μg.
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.
PCR Fragments
The DNA must be 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.

Host Strains
The host strain can have an impact on the quality of the template DNA prepared even using
the best methods.
DH5-α host strains consistently produce good results. HB101, XL-1 Blue, JM109 and MV1190
are usually fine. JM101 is not recommended.
The growth media you use can also affect the outcome yields, while LB is usually fine.
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 preferred method to quantitate DNA is to run an aliquot on a mini-gel and compare the
intensity to the control of a known concentration. There are also concentration ladders that
are commercially available. For each reaction, please provide at least 20ng/μl solution in
deionized water. Please provide at least 10μl additional sample for any possible re-
sequencing.
Macrogen strongly recommends Gel Electrophoresis rather than Nano-drop for
quantification.

2
Primers Preparation
Macrogen provides universal primers at no extra charge (see table below). If you prefer your
own custom primer, include your primer in a tube with your order at a concentration of 5-10
pmole/ μl=60 ng/μl, in deionized water at volume of greater than 20μl. A customer supplied
primer should be purified, or at least desalted. Crude primers generally do not work well for
sequencing.
Primer Considerations:
• High purity
• No secondary priming sites or mismatches
• Length 18-25 bases
• GC% content between 40% and 60%
• Tm (melting temperature) between 55°C and 60°C
• No significant hairpins (>3bp)
• Free of salts, EDTA, or other contaminants
Submitting Samples in an Individual Tubes
We recommend using a 1.5µl micro-centrifuge tube. Free re-sequencing is provided for
samples submitted in Individual tubes.

Submitting Samples in a 96-well Plate

We recommend placing your Template samples in a 96-well plate secured with 8-strip caps.
Identify on your order the universal primer from Macrogen’s library you wish to use or
submit with your plate of samples a tube containing your preferred custom primer.
The fee for sequencing an entire plate with the same primer is 1,045 NIS. The fee for using
multiple primers for the same plate is 1,558 NIS.
Samples supplied in a 96-well plate cannot be re-sequenced.
Please maintain as consistent a sample concentration as possible across the plate wells to
control well-to-well variation so as to optimize the sequencing results.

3
4
 Macrogen Supplied Universal Primers

Name Sequence (5'-3') Name Sequence (5'-3')

1492R TACGGYTACCTTGTTACGACTT M13R-pUC CAGGAAACAGCTATGAC

27F AGAGTTTGATCMTGGCTCAG MT_Forward CATCTCAGTGCAACTAAA

35S-A AAGGGTCTTGCGAAGGATAG pBacPAC-RP GTCTGTAAATCAACAACGC

35S-B AGTGGAAAAGGAAGGTGGCT pBAD-F ATGCCATAGCATTTTTATCCA

518F CCAGCAGCCGCGGTAATACG pBAD-FP ATGCCATAGCATTTTTATCC

800R TACCAGGGTATCTAATCC pBAD-R GATTTAATCTGTATCAGG

AD_Reverse AGATGGTGCACGATGCACAG pDONOR-FP TAACGCTAGCATGGATCTC

a-Factor TACTATTGCCAGCATTGCTGC pEGFP_N CCGTCCAGCTCGACCAG

AOX1_Forwar
GACTGGTTCCAATTGACAAGC pEGFP-FP TTTAGTGAACCGTCAGATC
d

AOX1_Rever
GCAAATGGCATTCTGACATCC pEGFP-RP AACAGCTCCTCGCCCTTG
se

BGH-R TAGAAGGCACAGTCGAGG pESP-RP TCCAAAAGAAGTCGAGTGG

GGGTTATGCTAGTTATTGCTCA
Bluescript_KS TCGAGGTCGACGGTATC pET-24a
G

Bluescript_SK CGCTCTAGAACTAGTGGATC pET-RP CTAGTTATTGCTCAGCGG

CMV-F CGCAAATGGGCGGTAGGCGTG pFastBac_Forward GGATTATTCATACCGTCCCA

CYC1_Rever pFastBac_Revers
GCGTGAATGTAAGCGTGAC CAAATGTGGTATGGCTGATT
se e

AGCTGGACATCACCTCCCACAAC
DsRed1-C pGEX3 GGAGCTGCATGTGTCAGAGG
G

DsRed1-N GTACTGGAACTGGGGGGACAG pGEX5 GGCAAGCCACGTTTGGTG

CGACTCACTATAGGGAGAGCG
EBV-RP GTGGTTTGTCCAAACTCATC pJET1_2F
GC

AAGAACATCGATTTTCCATGG
EGFP-C CATGGTCCTGCTGGAGTTCGTG pJET1_2R
CAG

EGFP-CF AGCACCCAGTCCGCCCTGAGC pMalE TCAGACTGTCGATGAAGC

EGFP-CR CGTCCATGCCGAGAGTG pQE-F CCCGAAAAGTGCCACCTG

EGFP-N CGTCGCCGTCCAGCTCGACCAG pQE-R GTTCTGAGGTCATTACTGG

EGFP-NR CGTCGCCGTCCAGCTC pREP-fwd_primer GCTCGATACAATAAACGCC

GAL1_Forwar
AATATACCTCTATACTTTAACGTC pRH_Forward CTGTCTCTATACTCCCCTATAG
d

CAAAATTCAATAGTTACTATCG
Gal4AD TACCACTACAATGGATG pRH_Reverse
C

GLprimer1 TGTATCTTATGGTACTGTAACTG pTrcHis_Forward GAGGTATATATTAATGTATCG

GLprimer2 CTTTATGTTTTTGGCGTCTTCCA QE_Promoter CCGAAAAGTGCCACCTG

5
 TAAACTTCAGGGTGACCAAAAAA
HCO2198 RVprimer3 CTAGCAAAATAGGCTGTCCC
TCA

ITS1 TCCGTAGGTGAACCTGCGG RVprimer4 GACGATAGTCATGCCCCGCG

ITS2 GCTGCGTTCTTCATCGATGC SP6 ATTTAGGTGACACTATAG

STag_18mer_Prim
ITS3 GCATCGATGAAGAACGCAGC GAACGCCAGCACATGGAC
er

ITS4 TCCTCCGCTTATTGATATGC SV40-pArev CCTCTACAAATGTGGTATGG

ITS5 GGAAGTAAAAGTCGTAACAAGG SV40-Promoter GCCCCTAACTCCGCCCATCC

ACCTACAACAAAGCTCTCATCAA
KAN2-FP T3 ATTAACCCTCACTAAAG
CC

GCAATGTAACATCAGAGATTTTG
KAN2-RP T7 AATACGACTCACTATAG
AG

GGTCAACAAATCATAAAGATATT ATGTCGTAATAACCCCGCCCC
LCO1490 T7_EEV
GG G

M13F GTAAAACGACGGCCAGT T7promoter TAATACGACTCACTATAGGG

M13-FP TGTAAAACGACGGCCAGT T7terminator GCTAGTTATTGCTCAGCGG

M13F-pUC GTTTTCCCAGTCACGAC U-19mer_Primer GTTTTCCCAGTCACGACGT

M13R GCGGATAACAATTTCACACAGG