UALR Biosciences Core Facility

High Throughput DNA Sequencing Fragment Analyses Real Time PCR In situ Hybridization Proteomics

High Throughput DNA Sequencing

Beckman CEQ 2000XL

DNA Analysis System

Beckman CEQ 2000XL DNA Analysis System

This is an automated unit capable of reading and analyzing DNA sequencing products that have been generated by incorporating WellRED fluorescent dyes into the DNA. The dyes fluoresce and are automatically detected and processed by the system’s software. The system is equipped with an array made up of eight parallel capillaries permitting electrokinetic injections of eight samples at a time. This high throughput setup allows for 96 samples to be processed and analyzed by the system's software in 24 hours. On average, sequences of 600 to 850 bases per sample can be generated, depending on primer and template qualities.

Amount of DNA Required

dsDNA 50 - 100fmol ssDNA 25 - 50fmol Purified PCR product 10 - 50fmol

Estimation of DNA Required

Size (kilobase pairs)

ng for 25 fmol ng for 50 fmol ng for 100 fmol

0.2 3.3 6.5 13 0.3 4.9 9.8 20 0.4 6.5 13 26 0.5 8.1 16 33 1.0 16 33 65 2.0 33 65 130 3.0 50 100 195 4.0 65 130 260 5.0 80 165 325 6.0 100 195 390 8.0 130 260 520 10.0 165 325 650 12.0 195 390 780 14.0 230 455 910 16.0 260 520 1040 18.0 295 585 1170 20.0 325 650 1300 48.5 790 1575 3150

For ssDNA, the values (ng) should be divided by 2.

Guidelines for DNA Sample Preparation***REMEMBER: HIGH QUALITY TEMPLATE WILL LEAD TO HIGH QUALITY DATA!!!!

IT IS TO YOUR ADVANTAGE TO FOLLOW PROCEDURE!!!!

Prepare templates properly:Resuspend in water or Tris-HCl. Do not resuspend in TE or EDTA reagents.Must be purified and free of ionic contaminants Best to use a commercial kit for purification!The Qiagen Qiawell and Qiaprep work well (dsDNA and ssDNA)Standard Alkaline Lysis Preps (NO PHENOL!!!)For PCR Products: Qiagen QIAquick PCR purification kit PCR products must be homogeneous

Quantitate DNA:SpectrophotometericFluorometeric Agarose Gel

Custom Primer Considerations:Quality primers necessaryPurified by desalting, HPLC or OPC purification preferredSize around 20 bases with 18 bases as a minimumGC content should be less that 50%Avoid primer dimerizationRatio for dsDNA is greater than 40:1 primer:template molar ratio (~3.2 ρmol).

Major Reagents/consumables

Sequencing Reaction

Sequencing Reaction

Sequencing Reaction

Capillary DNA Sequencing

1. Gel Fill

2. Sample Injection

3. Sample Separation

4. Sample Detection

5. Data Storage & Analysis

Data Analysis

Name   Length   Sequence

M13 (-21) Universal / Forward 17 mer 5’ GTA AAA CGA CGG CCA GT 3’

M13 Reverse Primer 18 mer 5’ CAG GAA ACA GCT ATG ACC 3’

T7 Promoter Primer 20 mer 5’ TAA TAC GAC TCA CTA TAG GG 3’

T3 Promoter Primer 18 mer 5’ TAA CCC TCA CTA AAG GGA 3’

SP6 Promoter Primer 18 mer 5’ ATT TAG GTG ACA CTA TAG 3’

Standard Primers

If you wish to supply your own primer we will require 5µl of 10pmol/µl primer.

Primer should be 18-24 bases in length and have a melting temperature (or Tm)

of 55ºC-60ºC. GC content should be 50-55%.

We offer the following standard primers at no additional cost.  

Sample Information SheetDate:____________ Time:____________

UALR Biosciences Core FacilityContacts: Nawab Ali, PhD., ETAS 426

Phone: 501-683-7340, Fax: 501-569-8039, Email: dnasequence@ualr.edu (DNA Sequencing Sample Sheet)

The Following must be filled out prior to processing. Please print.

User Name___________Address__________________Email__________________Phone #______________Principal Investigator________________Institution_______________Dept__________________UAR Account # ___________________BillingContact_____________________________Phone#__________

 

  

Sample Name Primer DNA Type(ss/ds/PCR)

DNA Concentration

(ng/µl)

DNA Size(including

insert)

Print OutY or N