How to prepare samples
Quantity to be sent
Form
PCR product purification service
Preparation of test tubes and compilation of the form
Samples shipment
Results
Red Box
White Box
To have good sequences you need to have good DNA. Here are some tips to prepare your samples for sequencing… and don't forget to ALWAYS elute in DISTILLED WATER!
Preparation of plasmid DNA:
• Use bacterial strains that do not recombine (DH5a, HB101, XL1 Blue etc.)
• Avoid contamination (Genomic, RNA, proteins, Salts etc. )
• Extract DNA with commercial kits with ion exchange technology or silica membrane absorption
• Eliminate contaminants that inhibit polymerase (EDTA, Ethanol, etc.)
• Elute in distilled water
• Quantify DNA with a spectrophotometer (260/280 greater than or equal to 1.8)
PCR Sample Preparation:
• Amplify as specifically as possible trying to consume all the oligos during the reaction and check the product obtained on agorose gel
• Avoid contamination (Genomic, RNA, proteins, Salts, etc.)
• Extract the DNA with commercial kits with silica membrane adsorption technology or possibly perform a gel filtration (G50 sephadex)
• If possible, avoid extracting DNA from the agarose gel
• Eliminate contaminants that inhibit polymerase ( EDTA, Ethanol, etc.)
• Elute in distilled water
• Quantify the DNA with the spectrophotometer (260/280 greater than or equal to 1.8)
Sample preparation for electrophoresis only:
If you want to do the sequence reaction yourself you must:
• Use the Big Dye Terminator V1.1 kit from Applied Biosystems (P / N 4337450) or V3.1 (P / N 4337455)
• Purify non-embedded samples (we recommend gel filtration with G50 sephadex)
• Use 0.2 ml tubes or 96-well microplates for shipping LYOPHILIZED samples
| PLASMIDIC | PLASMIDICO WITH REQUEST OUR OLIGO |
| Dna 800ng + Oligo 10pmoli = 20 µl Final volume. Up to 5Kb vector + insert (add another 100ng every 5Kb). |
Dna 800ng WITHOUT OLIGO = 15µl Final volume. Up to 5Kb vector + insert (add another 100ng every 5Kb). |
| PCR | PCR WITH OUR OLIGO REQUEST |
| DNA10 ng each 100bp of amplified + Oligo 10 pmoles=20µl Final volume. |
DNA 10ng every 100bp of amplified WITHOUT OLIGO = 15µl Final volume. |
| Up to 2000bp, contact the lab for fragments >2000bp | Up to 2000bp, contact the lab for fragments>2000bp |
| ADENOVIRUS | |
| Dna 1200 ng + Oligo 24 pmoles=20μl Final volume. |
If you do not have the requested quantity available, you can still send the minimum quantity (remember to check the minimum quantity box in the form) then:
| PLASMIDIC | PLASMIDE WITH OUR OLIGO REQUEST |
| Dna 400ng + Oligo 5pmoles = 10 µl Final volume. Fino a 5Kb vector +insert (add another 100ng every 5Kb). |
Dna 400ng WITHOUT OLIGO = 7.5 µl Final volume. Up to 5Kb vector + insert (add another 100ng every 5Kb). |
| PCR | PCR WITH OUR OLIGO REQUEST |
| DNA 6.5 ng per 100bp of amplified + Oligo 5 pmoles=10µl Final volume. |
DNA 6.5 ng per 100bp of amplified WITHOUT OLIGO = 7.5µl Final volume. |
| Up to 2000bp, contact the lab for fragments>2000bp | Up to 2000bp, contact the lab for fragments>2000bp |
| ADENOVIRUS | |
| Dna 600 ng + Oligo 12 pmoles=20μl Final volume. |
Form
To book the ride and to facilitate our work you should fill out the form in .xlsx format with your PC and send it by e-mail to the address: This email address is being protected from spambots. You need JavaScript enabled to view it.
The same form must be printed by reducing the set up to the filling of a single sheet and sent together with the samples.
Excel file ( .xlsx )
DOWNLOAD FORM SINGLE SAMPLES
Excel file ( .xlsx )
DOWNLOAD FORM FOR THE PLATES
PCR product purification service
The purification of the PCR products is carried out in high throughput using commercial silica membrane kits.It is important that the PCR product has only one band.
Material to be sent:
1) 50 ul of the PCR reaction in 0.2 ml tubes
2) Concentrated oligos 2 pmoles / µl (2 µM). The volume of oligo must be proportional to the quantity of reactions (volume = 6µl X no samples to be sequenced) and must be delivered in 0.2 ml tubes
3) Form filled in as per Bio-Fab indications:
- name the samples with surname and progressive number (starting from 01) and then add other information such as the name of the template and the oligo.
- First list all the samples to be purified and then in the queue those eventually to be sequenced with other oligos.
- If you need to sequence a sample with several oligos, you must fill in the form by putting all the samples to be sequenced first with the "first oligos" and, continuing the progressive numbering, add the names of the samples to be sequenced with other oligos, reporting in brackets the reference to the original sample, and therefore to the number of the test tube.
- Always indicate the length of the amplifiers.
4) The photo of the amplified products, 5 ul of the PCR, courses on agarose gel is strongly recommended. The size of the band and the size of the reference marker must be indicated.
Preparation of test tubes and compilation of the form
Remember to always follow these instructions:
- Send the samples in 0.2 ml pcr tubes;
- number the tubes on the cap and always in progressive order starting from 01, without repetitions and without skipping numbers. The numbering of the test tubes must correspond to the numbering shown on the form;
- Make sure that the tubes close correctly, it is advisable to wrap them with parafilm and insert them inside a Falcon tube;
- if you use the plates they must be oriented as shown in the figure, so sample n.1 must be at the bottom left and sample 8 at the bottom right on the short side:
- It is strongly recommended to use strips to close the wells and to wrap the plate in parafilm.
ATTENTION:
Samples that are not properly closed will risk leaking or evaporating during transport and will need to be re-shipped.>
When filling out the form we ask you to follow the following precautions:
- use the most updated version of our form that you can download at the bottom of the page;
- always indicate with an X the type of service requested (Sequencing Standard or Sequencing and nothing else);
- use a single form regardless of the type of template (PCR or plasmid), reporting any comments in the appropriate sections;
- always report the samples with surname and number (in order starting from 01, as well as the test tubes), all other information relating to the sample can be reported below. Example:
Samples shipment
The samples to be sequenced must be sent to this address:
Bio-Fab research srl
Via di Castel Romano, 100
Reception of goods, building 1 C
00128 ROME
BIOFAB COURIER SERVICE:
It is possible to take advantage of our sample collection service by courier at a cost of 9.00 euros for up to 30 samples, free for over 30 samples (even cumulative between multiple laboratories). To book a collection send an email to This email address is being protected from spambots. You need JavaScript enabled to view it. and indicate:
• Day of collection
• Time slot for collection
• Address and any additional information useful for the courier (floor, room, etc ...)
The samples must be inserted in a falcon or simply protected by a little bubble wrap to prevent the tubes from being damaged. Everything must be placed inside an envelope together with the relative form, no ice needs to be added. The bag must not exceed the weight limit of 1kg. At the same time as the booking confirmation of the collection, we will send you the waybill by email which must be printed and delivered to the courier at the time of collection.
As soon as the sequences are ready we will send you an e-mail (usually within 24 hours of receiving the samples). The files will be uploaded to your Reserved Area.
To open a new account just follow these simple instructions:
1. click on the RESERVED AREA button that you find at the top of all the pages of our site,
2. you will enter a new page: click on the button “Don't have an account? Register ",
3. fill out the registration form in its entirety,
4. registration completed! Now you can enter your Reserved Area. You will find the folders with the sequences in Laboratory results / Oligo Bank.
In order to open the sequence files we recommend the following SOFTWARE:
WINDOWS ENVIRONMENT:
Compatible with Windows XP, Vista, 7, 8, 10
Chromas, available in freeware version:
www.technelysium.com.au/chromas.html
Abiview !: download Abiview in zip format
OTHER PLATFORMS (PC, Mac, Linux)
We recommend: TRACE VIEWEW at this address: www.codoncode.com/TraceViewer
SEQUENCE COLLECTION POINT (RED BOX)
For customers in Rome and surroundings a sample collection point is active at the DEPARTMENT OF MOLECULAR MEDICINE, SAPIENZA UNIVERSITY OF ROMA – avenue Regina Elena, 291 - with free collection on Monday - Wednesday - Friday.
INSTRUCTIONS FOR DELIVERY OF SAMPLES (RED BOX):
POINT FOR THE OLIGO RETREAT (WHITE BOX)
The box for the collection of oligos (WHITE BOX) is located in the main entrance of the General Physiology Building (Department of Biology and Biotechnology) SAPIENZA UNIVERSITY of ROMA with free daily collection.
The access time to the box is from 8.30 to 18.00
INSTRUCTIONS FOR OLIGO WITHDRAWAL (WHITE BOX):
