DNA Sample preparation and Submission Guidelines

Transcription

1 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 Ready Read (Linking) Value Read Plasmids Comfort Read plasmids PCR products Primers 1 Amount Required* Post sequencing purified products in Hi-Di formamide 1-2µg dried 2-3µg dried 10ul 0f 20-50ng/ul for PCR products<500bp 10ul of ng/ul for PCR products>500bp Shipment at 4 o C or lower is recommended 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.

2 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, 1 st floor, EPIP 2 nd phase, Whitefield, Bangalore Delivery of Results: DNA Sequencing results are sent by 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 = (0.41 x GC %) (650/length) Calculate the average Tm of primer pair and choose an annealing temperature that is 3 o 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

3 Eurofins recommends shipment of PCR products at 4 o C 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 30 o C 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

4 For primer walking details, please mail us at BAC, PAC, YAC and cosmid sequencing: Cosmids: Send cosmid-dma dissolved in water. Recommended template concentration: 0.5µg/µl H 2 O. 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, 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

5 HPSF is a registered trademark of Eurofins. Optimal Primer Concentration: 10pm/ µl. Required volume is µl. Be aware that primers to be utilized for sequencing will need to have as close to 50% GC content as possible, and be 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 bases. Each primer should have GC content of percent. There should not be more than three identical contiguous bases in a given sequence. The primer annealing temperature should between o Celsius. 5