Jun 11, 2026
Whole-Plasmid Sequencing Send-out (Plasmidsaurus)
- Raven Coffer1,
- Owen Butler1
- 1DAMP Lab
- DAMP Lab
Protocol Citation: Raven Coffer, Owen Butler 2026. Whole-Plasmid Sequencing Send-out (Plasmidsaurus). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbjo8yvpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2026
Last Modified: June 11, 2026
Protocol Integer ID: 314357
Keywords: plasmidsaurus for sanger sequencing, submission of plasmid dna sample, plasmid dna sample, plasmidsaurus guideline, plasmidsaurus, plasmid sequence, correct the plasmid sequence, plasmid, sanger sequencing, sequencing result
Abstract
This protocol outlines the preparation and submission of plasmid DNA samples to Plasmidsaurus for Sanger sequencing. Samples are submitted according to Plasmidsaurus guidelines, and the sequencing results are used to verify correct the plasmid sequence and construct integrity.
Materials
Consumables
- Filtered pipette tips
- Microcentrifuge strip-tubes
- 50 mL conical tube
- KimWipes
- Small bag
Equipment
- Pipettes
- Vortexer
- Benchtop centrifuge
Reagents
- Nuclease-free water
Before start
- Determine the concentration and purity of the samples. DAMP Lab recommends to use Qubit for DNA quantification, as it is more accurate than nanodrop, particularly at lower concentrations.
- Ensure the samples meet the quality control metrics necessary for successful sequencing results:
A260/A280 ratio of ~1.8
A260/A230 between 2.0-2.2
Preparation
13m
Setup
Using Table 1, choose the appropriate service for your samples, based on the plasmid size and concentration.
| Plasmid Size | Sample Concentration (ng/uL) | Order Name | Required Volume (uL) | |
| < 25 kb | 20 - 200 | Standard Low Concentration | 10 | |
| < 25 kb | 200 - 1000 | Standard High Concentration | 4 | |
| 25 - 125 kb | 50 - 400 | Big | 20 | |
| 125 - 300 kb | 50 - 400 | Huge | 40 |
Table 1. Plasmidsaurus plasmid sequencing sample requirements (2026)
Note
DAMP Lab generally sequences plasmids < 25 kb, thus we usually select Standard Low (or) High Concentration sequencing. When deciding between Low or High Concentration options, use the following guidelines:
- if all samples are greater than 200 ng/uL, select High Concentration
- if any samples are less than 200 ng/uL, select Low Concentration
1m
Download the spreadsheet below to calculate the dilutions for each sample. The following steps will be completed on the spreadsheet.
TEMPLATE_Plasmidsaurus_SEQ.xlsx10.7KB Part 1. Highlight the correct sequencing type.
The correct sequencing type was determined in Step 1.1, using Table 1. DAMP Lab recommends to dilute all samples to values slightly above the lower end of the accepted sample concentration range. This will save valuable sample for downstream applications, and ensure that there is sufficient overage for successful sequencing. Similarly, DAMP Lab recommends to send slightly more volume than is necessary, to avoid sequencing issues. Table 2 (which is reproduced in Part 1 on the spreadsheet) outlines the DAMP Lab recommended values.
(ACTION) On the spreadsheet, highlight the corresponding row for the pre-determined sequencing sample type.
| Sample Type | Recommended Concentration (ng/uL) | Recommended Volume (uL) | |
| Standard Low Concentration | 30 | 15 | |
| Standard High Concentration | 250 | 6 | |
| Big | 100 | 25 | |
| Huge | 100 | 45 |
Table 2. DAMP Lab recommended concentrations and volumes for Plasmidsaurus sequencing.
Part 2. Fill in the final concentration and volume to be sequenced.
(ACTION) On the spreadsheet, fill in the grey boxes with the recommended concentration and volume, as determined by the sample type and corresponding highlighted row in Part 1 (which are the same as in Table 2).
Part 3. Fill in the sample concentrations.
(ACTION) On the spreadsheet, input the sample IDs and concentrations into the yellow boxes.
If some samples fall below the recommended concentration for your chosen sequencing type, and are above the minimum required concentration, they can still be sent for sequencing successfully. In this case, do not dilute the samples at all, but still send the DAMP Lab recommended volume. (ACTION) For these samples, manually edit the row of the affected sample in Part 3 to show that 0 uL of nuclease free water were added (blue box), and the recommended volume of sample (purple box) was equal to the recommended volume for the respective sequencing type.
** Note that the placement of the sample in the spreadsheet automatically corresponds to a Sample Number, which determines the location of the sample in the microcentrifuge strip-tube. Consistent ordering is critical for correct sample assignment. **
Note
For example: A set of samples (containing plasmids >25 kb) includes the following concentrations (ng/uL): 23, 30, 39, 60, 130.
Part 1: You would have previously selected Standard Low Concentration as your sample type (based on Table 1). Highlight row 5 to indicate that you choose Standard Low Concentration sequencing.
Part 2: Fill in the grey boxes with 30 ng/uL for the concentration, and 15 uL for the volume.
Part 3: Fill in sample names and concentrations. For the sample with a concentration of 23 ng/uL, manually change the volume of nuclease free water = 0 uL (blue box), and the recommended volume of sample (purple box) = 15 uL.
5m
Save the edited spreadsheet, and upload it to the appropriate location for record-keeping and future reference.
1m
Login to the DAMP Lab account on Plasmidsaurus. Select "Plasmid" and choose the type of sequencing, determined in Step 1.2. Input the order name which includes the date, project, and your initials. Copy the sample names from the spreadsheet, and paste into the sample names box on Plasmidsaurus. Click review order.
Locate the closest Plasmidsaurus dropoff box, using this map. (DAMP Lab usually selects #573.) Choose Dinocoins as the payment option, and ensure there are sufficient Dinocoins to available for the order. Notify the Lab Manager if the Dinocoins balance is insufficient. Under the Share Results section, enter the emails of the personnel relevant to the project. Click Place order.
Print the order form.
3m
Label
Number the microcentrifuge tubes starting at "1" and ending with the number of samples to be sequenced. Label tube #1 (and the first tube of each additional microcentrifuge tube, if required) with the first 2 letters of the order code, as shown in Figure 1.
Figure 1. Microcentrifuge tube labeling. Image from Plasmidsaurus.
3m
Procedure
7m
Sample Preparation
Dilute the samples. The spreadsheet will have calculated the amount of water and sample required to dilute each sample individually to the recommended concentration. First, add the correct volume of nuclease-free-water to each tube. Second, add the correct volume of the respective sample to each tube.
Ensure that the sample is added to the correct tube listed on the spreadsheet.
5m
Securely close caps securely on the microcentrifuge tubes. Vortex for 3-5 seconds. Spin down using a benchtop centrifuge for 5 seconds.
Sample Submission
Put the microcentrifuge strip-tubes into a 50 mL conical tube with a few kimwipes for padding.
30s
Place the conical tube into a bag which contains the order sheet, folded such that the QR code is visible. Seal the bag securely.
30s
Place the sample to the correct dropbox (which was determined in Step 1.4) prior to the daily pickup time.
1m
Cleanup
3m
Storage
Return samples to the proper storage location.
1m
Decontamination
Wipe benches and pipettes with DNAaway, RNase Aaway. Afterwards wipe benches and pipettes with 70% ethanol.
2m
Protocol references
Acknowledgements
Drafted by: Raven Coffer
Uploaded by: Luciana Lozano
Edited by: Raven Coffer, Owen Butler, Kristen Sheldon, Lena Landaverde, Molly Brennan