Download DNA submission instructions - Eurofins Genomics India Pvt Ltd

DNA Sample preparation and Submission Guidelines
Requirements:
•
Please submit samples in 1.5ml microcentrifuge tubes.
•
Fill all the required information in the Eurofins DNA sequencing order form and
send a soft copy to [email protected]
•
Send gel picture of the samples run on a agarose gel to determine the quality and
the quantity of the products.
•
If you have more than 50 samples, please send samples in 96-well format to
expedite delivery of results.
•
Assign template names to 8 characters or less. Assign primer names at 15
characters or less.
•
Please do not use Tris EDTA buffer for eluting/dissolving your samples.
•
Eurofins will store left-over samples for up to 1 month. If you need these to be
returned to you, please indicate the same in your submission form under
additional instructions. Do note that you will incur additional delivery charges for
the same.
Quick Reference Chart:
DNA Type
Amount Required*
Ready Read (Linking)
Post sequencing purified products in Hi-Di formamide
Value Read Plasmids
1-2µg dried
Comfort Read plasmids
2-3µg dried
PCR products
10ul 0f 20-50ng/ul for PCR products<500bp
10ul of 50-100ng/ul for PCR products>500bp
Shipment at 4oC or lower is recommended
Primers
15-20µl of a 10µM solution(10pmol/µl).
* Our lab is optimized to run the samples meeting these conditions. Failure to send the samples
within the above requirements may lead to an increased number of failed reactions, slower
processing time and incur additional failure rate costs.
1
Shipment and Packaging:
Purified DNA, plates may be sent at room temperature. Purified DNA should be provided
in double distilled water. Never use TE. Glycerol stocks should be sent on dry ice.
Provide adequate padding to prevent tubes from being crushed during shipment. Seal
caps with Para film or plastic wrap to keep them from opening.
Ship the samples to:
Eurofins Genomics Pvt Ltd
DNA sequencing Lab,
#183, Gayathri Tech Park,
1st floor, EPIP 2nd phase,
Whitefield, Bangalore – 560 066
Delivery of Results: DNA Sequencing results are sent by e-mail to the id mentioned in
the order form. For large number of samples (15-20 plates in 96-well plate format)
alternative methods of results delivery by FTP transfer or CD/DVD is possible.
PCR Products:
Optimization of the PCR Reactions:
•
Calculate the melting temperature TM of your primers with the formula:
Tm = 69.3+ (0.41 x GC %) – (650/length)
•
Calculate the average Tm of primer pair and choose an annealing temperature that
is 3o C below the average Tm.
•
Do not apply more than 10pM of primer for 50ul reaction
•
Add no more than 1% end concentration of DMSO or Tetramethylammonium
chloride (50mM end concentration) when protocol requires their use. Use only
ultra pure chemicals (SIGMA, Molecular Biology grade).
•
Your quality check should reveal only one band. Sequencing results are highly
dependent upon the quality purification of your PCR products.
2
•
Eurofins recommends shipment of PCR products at 4oC or lower temperatures.
We have found that this additional precaution results in a higher success rates and
good quality data.
Template preparation:
Please send column or membrane purified PCR products. However, we can do the
purification process at our facility for a nominal charge per sample if needed. Do not
lyophilize your PCR products because this will result in poor sequence results. We do
not recommend Gel Extraction methods for PCR purification.
Plasmid DNA:
E.Coli strains and cultivation
An essential requirement for excellent sequencing results from ds-DNA is the use of a
recA and endA E.Coli strains (DH5ά, XL1-blue, JM109 etc,). From our experience the
strain DH5ά gives very high yields of quality DNA. Plasmid preparations from stationary
cultures are not optimal. If you have to cultivate your bacteria at 37 o C, please measure
the OD and harvest the cells at an OD600 of 1.5. Alternatively, bacteria can be grown at
30oC overnight and harvested without OD measurements.
DNA Isolation
Recommended purification methods are Nucleospin prep, Sigma, Promega, QIAGEN
and Axygen column kits. We do not recommend phenol-chloroform method. Elute the
DNA in water and check the integrity of the plasmids via agarose gel electrophoresis. Be
sure to send us a photo of the gel along with your samples. After elution of the DNA
from the column please check the amount of DNA and provide concentration of DNA.
Provide the dried pellet in a 1.5ml tube. If you have large number of samples (more than
50), submit your samples in 96 well plates for faster automation purposes.
Amount of DNA Required
Value read sequencing: 1-2µg
Comfort Read Sequencing: 2-3 µg
3
For primer walking details, please mail us at [email protected]
BAC, PAC, YAC and cosmid sequencing:
Cosmids:
Send cosmid-DMA dissolved in water. Recommended template concentration: 0.5µg/µl
H2O. We typically utilize 1.5µg per reaction. To allow for trouble shooting and repeat
reactions, we recommend 6µg of material.
For BAC, PAC, YAC details, please mail us at [email protected].
Primers:
Eurofins will utilize HPSF® purified standard vector primers for sequencing. You may
also send your own specific primers for sequencing. However, we highly recommend that
you provide HPSF® purified oligos from Eurofins. Our sequencing laboratory routinely
sees an increase in sequencing quality due to decreased background and longer read
lengths when oligos are supplied from Eurofins. For a listing of all primers not mentioned
below, email us at [email protected].
Vector primer
Sequence
M13 Reverse (-29)
CAG GAA ACA GCT ATG ACC
M13 Forward (UNI)
TGT AAA ACG ACG GCC AGT
M13 Forward (-43)
AGG GTT TTC CCA GTC ACG ACG TT
M13 Reverse (-49)
GAG CGG ATA ACA ATT TCA CAC AGG
SP6
CGA TTT AGG TGA CAC TAT AG
T3
AAT TAA CCC TCA CTA AAG GG
T7
TAA TAC GAC TCA CTA TAG GG
T7 Terminator
CTA GTT ATT GCT CAG CGG T
pBAD Forward
ATG CCA TAG CAT TTT TAT CC
pBAD Reverse
GAT TTA ATC TGT ATC AGG
pET Upstream
ATG CGT CCG GCG TAG A
pGEX Reverse
CCG GGA GCT GCA TGT GTC AGA GG
pGEX Forward
GGG CTG GCA AGC CAC GTT TGG TG
BG Reverse (PCR3.1)
TAG AAG GCA CAG TCG AGG
4
HPSF® is a registered trademark of Eurofins.
Optimal Primer Concentration: 10pm/ µl. Required volume is 15-20 µl. Be aware that
primers to be utilized for sequencing will need to have as close to 50% GC content as
possible, and be 19-21 bases in length. Primers that do not meet these specifications will
not generate high quality results and could result in higher failures and additional costs.
Tips for primer design:
If you wish to design your own sequencing primers, optimal length is between 18-22
bases. Each primer should have GC content of 50-60 percent. There should not be more
than three identical contiguous bases in a given sequence. The primer annealing
temperature should between 50-65o Celsius.
5