Template and Primer Requirements
Sample/Primer Volume
If using CCG primers, provide template in total volume of 10 ul H2O.
If using your own primer, provide template with 10-15 pmoles of primer in a total volume of 14 ul H2O.
Template Requirements:
Template Quantity
PCR products
100-200 bp 2.5 – 7.5 ng
200-500 bp 5.0 – 25 ng
500-1000 bp 12 – 50 ng
1000-2000 bp 25 – 100 ng
> 2000 bp100 – 250 ng
As a general rule for PCR fragments, use 2.5 ng/100 bp of amplified product.
Plasmids <20 kb 400-1000 ng
Template purity is critical for sequencing.
Plasmids should be mini-prepped with a commercial column.
As a general rule for plasmids, use 100 ng/1kb plasmid.
Cosmids, BAC and templates > 50 kb1 g template
Please make sure to indicate on the form, the nature of the template as the sequencing formula differs for these templates.
Quantify DNA by Spectrophotometer or Fluorometer
The CCG lab provides access to a Nanodrop Spectrophotometer. This unit allows for the accurate determination of DNA concentration using as little as 1.5 ul of sample. The instrument provides a full spectral-trace from 230-350 nm. Pure DNA should give an OD260/280 of between 1.7-1.9 (1.5-1.7 is usually OK) and an OD260/230 of about 2.0. Low 260/280 indicates protein contamination, high OD260/280 indicates possible RNA or residual organics contamination. Low OD260/230 indicates contamination by organics and/or salts.
Effect of Too Little Template
Too little template or primer reduces the signal strength and peak height. In the worst case, the signal- to-noise level decreases so that bases cannot be called.
Effect of Excess Template
Excess template can affect data quality when present in sample loading onto the DNA Analyzer. Excess template inhibits the injection of extension fragments thus affecting signals generated from the instrument. Excess template can behave similarly to proteins and accumulate in the capillary array, which affects data resolution.
Template Quality
Please provide samples in an EDTA-free solution.
If significant secondary structure is suspected in a template, let us know on the Sequence Submission Form; we have a chemistry available that often resolves issues related to
structure (please note, use of this chemistry increases the likelihood of compressions early in the sequence.
Please see the notes below for additional comments on Template Quality.
Primers:
CCG Sequencing Primers:
GW1GTT GCA ACA AAT TGA TGA GCA ATG C
GW2GTT GCA ACA AAT TGA TGA GCA ATT A
M13F (-20)GTA AAA CGA CGG CCA GT
M13R (-27)CAG GAA ACA GCT ATG AC
SP6TAC GAT TTA GGT GAC ACT ATA G
T3AAT TAA CCC TCA CTA AAG GG
T7TAA TAC GAC TCA CTA TAG GG
T7 – shortTAA TAC GAC TCA CTA TAG
T7 - pCS2+AAT ACG ACT CAC TAT AG
T7 – terminatorGCT AGT TAT TGC TCA GCG G
Using Core Primers: in the “Core or Own Primer” column of the Sequence
Submission Form, simply note the primer you’d like us to usewith your sample,
for example, “T7”, “SP6”, etc.
Your Own Primer:
Length of 18-25 bases
GC content of 40-60%
Tm of 50-60oC
No secondary priming sites
No dimerization capability
No significant hairpins (> 3 bp)
Using Your Primers: in the “Primer” column of the Sequence SubmissionForm, you may either leave this blank, type “own”, or for record-keeping purposes, type the name of your primer.
If you have added a primer named the same as a primer available through theCCG, you may still list it in the “Primer” column on the Submission Form, but please, to prevent our adding a second aliquot of the primer make sureyou clearlyindicateyou have added the primer.
Template Quality
The 3730 is a capillary based DNA analyzer that uses electrokinetics for injection of samples into the capillaries. Therefore your samples must beSALT FREE AND PROTEIN FREE.Salt and protein are preferentially injected over DNA and also plug up the capillaries.
Do NOT dilute or resuspend the DNA in TE.Use ddH20 or 10 mMTris pH 8.5.
If using a column-based kit, insure that all of the EtOH is evaporated before eluting the DNA.
If concentrating the DNA insure that all of the EtOH is evaporated before re-suspending the DNA.
When using Qiagenminiprep kits, perform the PB wash regardless of the cell line used.
Effect of Residual Salts
The 3730 DNA Analyzer is especially susceptible to salt in samples from template preparation. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths. If salts and unincorporated dyes are not removed from the sequencing reaction, they will compete with extension fragments during electrokinetic injection and result in weak signals.
Effect of Proteins
Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array adversely affecting data resolution.
Effect of Residual Detergents
Some methods of phage template preparation use detergents such as Triton X-100. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.
Effect of Residual RNA
Residual RNA that is present in DNA template preps competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.
Frequent reasons for failure to get good data
Failure results when there is an insufficient level of fluorescent termination products for the computer software to assign a sequence. Some possible reasons:
- The 3730 capillary sequencer provides longer reads but is also more sensitive to residual salt and ethanol in sample. If you are experiencing variable sequencing results try an ethanol PPT or elute in water instead of the supplied Elution Buffer.
- Too little template results in reactions with little or no signal and poor or no base calling.
- Too much DNA produces reactions that terminate prematurely, often with fewer than 250 bases of reliable sequence data.
- Poor quality template DNA. Template DNA must be free of residual ethanol and salt.
- Insufficient primer concentration or poor quality.
- The Tm of the primer is < 50 C.
- The template does not contain a sequence complementary to the primer.
- Primer and/or template was not added to the reaction.
- Using the same primers for sequencing as were used for PCR.
- Samplecontaining plasmids from two or more different colonies was submitted inadvertently. CFU’s from densely plated colonies may contain a mixed population of plasmids. This results in two or more overlapping sequences on the electropherogram.
- Sample containing two or more PCR products was submitted inadvertently. PCR fragment was not gel purified or during gel purification the region of theagarose gel from which a PCR fragment is excised and eluted contains a mixed population of fragments.