Recommended DNA purification kits
The CMMT Stores has a selection of DNA purification kits in stock
- Applied Biological Materials DNA Purification Kits
- Qiagen DNA purification kits
- Other purification kits may be suitable
SAMPLE PREPARATION NOTES
Whole Plasmid Sequencing – NEW
Sample requirements
- Purification: use a commercial kit (see recommendations above)
- Buffer: resuspend in dH₂O or 10 mM Tris pH 8.0–8.5 (not TE)
- Submit samples in 8-strip PCR tubes or plates
- Email your sample Sample Submission form when submitting samples
- Quantity and concentration requirements:
| Plasmid Size | Minimum Quantity for Submission |
Minimum Concentration |
| <6 kb | 200 ng | 20 ng/ul |
| 6-10 kb | 350 ng | 30 ng/ul |
| 11-15 kb | 500 ng | 40 ng/ul |
| 16-25 kb | 850 ng | 80 ng/ul |
| 26-50 kb | 1500 ng | 100 ng/ul |
| 51-100 kb | 3000 ng | 250 ng/ul |
Deliverables
- Annotated plasmid map
- Full-length assembly (FASTA, FASTQ)
- Validation report (epi2me clone validation workflow) including:
- Per-base quality metrics
- Assembly statistics
- Sample output data (pBluescript) can be downloaded from our Google drive. Open the wf-clone-validation-report.html to view the report including plasmid map.
Applications
- Confirm plasmid integrity and structure
- Verify cloning experiments and insert orientation
- Detect mutations, rearrangements, or unexpected sequence changes
Pre-mixed Sample Service (Sanger Sequencing)
Special pricing is available for plasmid and PCR products if the following requirements are met:
- Primers must be pre-mixed with your plasmid or PCR product samples.
- Aliquots of standard primers stocked by the facility may be picked up in advance.
- Samples must be submitted in 8-strip PCR tubes or 96-well plates
Concentration and volume requirements
| Sample type | DNA Length (including vector) | DNA Amount (ng) | Primer Amount (pmol) | Total Volume (ul) |
| Plasmids | < 6kb | 300 | 10 | 15 |
| 6-10kb | 400 | 10 | 15 | |
| >10kb | 500 | 10 | 15 | |
| PCR Products | 100-200 bp | 20-30 | 10 | 15 |
| 300-500 bp | 40-60 | 10 | 15 | |
| 600-800 bp | 70-90 | 10 | 15 | |
| 1-2 kb | 120-160 | 10 | 15 |
Standard Service (Sanger Sequencing)
General Requirements
-
Purify templates using commercial kits (Qiagen recommended)
-
Normalize all samples to the same concentration
-
Provide concentration values for each sample
Minimum quantities and concentration ranges
| Sample Type | Minimum Quantity required per reaction |
Minimum Quantity required for submission |
Concentration Range1 |
| Plasmid | |||
| <7 kb | 160 ng | 320 ng | 25-160 ng/ul |
| 7-8 kb | 170 ng | 340 ng | 26-170 ng/ul |
| 9-10 kb | 180-200 ng | 360-400 ng | 30-200 ng/ul |
| 11-15 kb | 200-300 ng | 400-600 ng | 46-300 ng/ul |
| PCR Products | |||
| 100-200 bp | 10-15 ng | 20-30 ng | 2.3-15 ng/ul |
| 300-500 bp | 20-30 ng | 40-60 ng | 4.6-30 ng/ul |
| 600-800 bp | 35-45 ng | 70-90 ng | 6.9-45 ng/ul |
| 1000-2000 bp | 60-80 ng | 120-160 ng | 12.3-80 ng/ul |
- $3/reaction surcharge if concentrations must be adjusted
- $1.50/reaction surcharge for BAC/cosmid or low-concentration samples
- Resuspend in dH₂O or 10 mM Tris pH 8.0–8.5 (not TE)
- Standard primers provided; non-standard primers at 3.2 pmol/µl, at least 4ul per reaction
Ready-to-Run Service
- Provide cycle-sequenced and purified sequencing products
- Submit in 8-strip PCR tubes or 96-well plates if >6 samples
- Facility will run samples, confirm results, and provide data in .ab1 or requested format
Protocol References
- Usage of the Oxford Nanopore Clone Validation workflow
- Sequencing reaction protocol for BigDye: BigDye Terminator v3.1 Cycle Sequencing Kit Protocol (Applied Biosystems protocol)
- ThermoFisher Connect online Data Analysis Apps. Click here to connect.
- Finch TV to view, edit, print, and blast your sequence data.
- ApE-A Plasmid Editor, to view, edit, and analyze data, including alignment of multiple sequences.
- Sequence Analysis Sofware from ABI to edit and view both analyzed and raw sequence data, clear range at 5’ and 3’ ends, QVs and QV bars for the sequences, and export traces in multiple formats including pdf, phd.1, seq etc.
Feedback
If you have any suggestions for additions or edits to this page, please contact the facility.
Return to DNA Sequencing Core Facilities main page.