PCR Product Purification

The MGH DNA Core offers a small-scale (10µl/well) PCR purification that is intended for sequencing only. PCR products can be submitted to our facility directly following the amplification reaction. This service is intended for the extraction of the desired amplified PCR product, removing residual enzyme, salts, and primers. The isolated fragments are then processed for DNA Sequencing.

If you would like a large-scale purification for other downstream applications, clickhere.

Guidelines (for small-scale PCR purification):
  • Plates submitted for this service will be purified and immediately entered into the sequencing queue.
  • Samples must be submitted in a sturdy, half skirted 0.2mL 96 well plate. The plates can be purchased from USA Scientific. (USA Scientific catalog # 1402-9700)
  • Each well must contain 10µl of sample.
  • PCR purification is automated. For this portion of the service there is a flatrateper plate.
  • Fill the plate A1 → A12, B1 → B12, etc. (across, then down). Do not leave empty wells in between samples.
  • Be sure that your order form matches the contents of the plate.
  • A tube containing 400µL of sequencing primer at 10ng/µl must be submitted with the plate.

Sample Preparation

DNA Templates

Templates should be free of salts and other contaminants. Most commercially available DNA preparation kits will produce adequately-pure template if protocols are followed with care. Avoid over-growing of cultures. Best results are obtained with DNA samples having a 260/280 OD ratio of 1.8 or greater. DNA samples should be diluted in dH2O to the following concentrations:

Double-stranded plasmids: / 0.1 to 0.2 µg/µL
Cosmids or lambda clones: / 0.4 µg/µL
Single-stranded phage: / 0.1 µg/µL
PCR products: (less than 400bp long) / 20 ng/µL per 100 base pairs of product
PCR products: (over 400bp long) / Calculate your concentrations as you would for a plasmid.

PrimersPCR Primer Design Guidelines

Primers must be submitted mixed in the same tube or well with template DNA at a final concentration of 10ng/µL. For best results, primers should be designed with the following in mind:

  • Primers should be between 18-24 nucleotides in length
  • Primers should have a calculated Tm of greater than 50°C but less than 65°C
  • Avoid runs of a single base (e.g. 3 to 4 repeats of the same base)
  • Avoid primers with secondary structures or the potential to self-hybridize

Examples of standard pre-mixed reactions

For Double Stranded Plasmid DNA:

  • 10µL of DNA template at a concentration between 0.1µg/µL and 0.2µg/µL
  • 10µL of Primer at a concentration of 10ng/µL
  • Final volume will be 20µL.

For PCR Products (less than 400bp):

  • 10µL of DNA template at a concentration of 20ng/µL per 100 base pairs of product added to:
  • 10µL of Primer at a concentration of 10ng/µL
  • Final volume will be 20µL

Sample Submission Guidelines

Tube format

  • Please use only standard 1.5ml flip-cap "Eppendorf" type tubes.
  • NOT acceptable: 0.2ml tubes, 0.5ml tubes, strip-tubes, screw-cap tubes (unless shipping via air mail)
  • Label the tubes on the top/lid as clearly as possible.
  • Whether mailing or dropping off, tubes should be submitted in order as listed on the submission form (not randomly placed, and not loose/unracked).
  • If using local drop-off points, please rack tubes in the boxes provided and attach order form with rubber band. If no boxes available, send us a note, and submit as below:
  • If shipping your order or if no boxes available, please tape tubes to order form (or attached sheet) in order as listed on submission form. You may also use any rack or box designed to hold 1.5ml tubes.
  • Delays!Problems with tube orders are the most frequent cause of delays. Please prepare your order with care, and we will process it as quickly as we can.

Plate format

96-well plate format is acceptable and even preferred, as long as the following guidelines are followed:

  • Use only V-bottom plates with water-tight seals
  • Fill in your plate proceeding from A1 to A12, B1 to B12, etc. (across, then down). The maximum allowable number of samples is 95 per plate. You MUST leave plate position H12 empty.
  • The plate does not have to be full, but do not leave empty spaces between samples on the order form.
  • The order form must match the order and position of samples in the plate.
  • Primer and template should be pre-mixed in the plate (same as in tubes) to a total volume of 20µl
  • Only full plates (95 samples on the order form) qualify for thelower full-plate rate.
  • Plates forPCR purificationare subject to further restrictions.

Results / Turnaround Time

Our goal is 24-hour turnaround for sequencing samples received by noon Monday through Friday. Samples received after noon will be refrigerated until the following work day. Fast and efficient turnaround is only possible if orders are submitted in proper format.

Sequencing results are posted on our website following the completion of each run cycle. They may appear at any time of day. An email will be sent when your order is ready for download. On the download site, there will be two files:

  • ORDER#.DATE.seq contains all sample sequences concatenated (one after the other) in text format, uncompressed. Copypaste the contents to any text editor.
  • ORDER#.DATE.sit is a compressed file containing all the chromatogram/traces and individual text sequence files. When decompressed it will create a folder containing 2 files per sample:
  • SAMPLENAME.ab1 is a chromatogram file which can be viewed with a number of software applications (see below for suggestions).
  • SAMPLENAME.seq is a text file.

We strongly recommend that you download your data to your own computer and a secure archive location. The DNA Core does not keep a long-term data archive. The text version of your sequences may be enough for your purposes, but if you want to visually inspect trace curves, make basecalling edits, perform detailed alignments, etc. then we suggest that you open and view the chromatogram (*.ab1) files. Here are links to some free software that you may find useful. There are other programs available for purchase:

Decompression Software / CHromatogramChromatogramViewers / Windows to Mac Conversion Additional Software Required
PC / StuffIt Expander / Chromasor
BioEdit / N/A
Mac / StuffIt Expander / Edit Viewor
4Peaks / Windows to Mac conversion

Follow the steps below for setting up the required software for chromatogram viewing:

  1. Click onSequence Results.
  2. Click on the *.sit file. We suggest saving to your computer, then find it and open it with Stuffit Expander. A folder will be created with all *.ab1 and *.seq files inside.
  3. To view the *.seq files, you can right click and open with any text editing software (Word, Notepad, etc.). You may also change the file extension from *.seq to *.txt.
  4. To view the *.ab1 files you will need chromatogram viewing/editing software. If you are using a Mac, you may need ABI's3100 Conversion Utilityto prepare files for viewing with EditView, etc.

Difficult Template Sequencing

The MGH DNA Core Facility offers an alternative protocol to increase the read length of samples containing hairpin structures, GC-rich regions, AT-rich regions, areas containing various repeats, homopolymers regions and RNAi templates. There is an additional charge for this option.

For information about pricing for difficult template sequencing, click on the Rates link at the top of this page.

Range & Accuracy

High quality DNA templates generally yield sequence read lengths of up to 700 base pairs, with a cumulative error rate generally less than 1% over the first 400bases. Range and accuracy are critically dependent on the purity and quality of the DNA and template-primer compatibility.

Rates

Pricing effective Jan. 1st, 2013. All prices reflect actual cost of operation as required by Partners Research non-profit guidelines. Due to Partners and NIH regulations, rates are not the same for all users. Click one of the options below for rates:

  • Partners HealthCare Members
  • Academic and Medical Centers
  • For-Profit Organizations

Possible Delays

  • Delayed sample results:
  • If your results are delayed, please check Order Status. If it is stuck onReceivedfor a long time, then you should contact us. (Also see below).
  • Weekends and Holidays:
  • The Sequencing group operates at a reduced capacity over the weekend. Samples received on Friday before noon will most likely be finished by Sun afternoon, but not later than Monday afternoon - unless Monday is a holiday. The DNA Core follows the MGH holiday schedule.
  • Tips for avoiding delayed results:
  • Problem orders are processed last. Common causes of this include: tubes out of order, illegible handwriting on samples, wrong page of submission sheet printed, order not confirmed. We will do our best with problematic orders, but if we cannot match your tubes to your order form, then we will have to cancel your order or risk returning mismatched data. Also, please be sure to give us a phone number at which you can actually be reached so we can contact you to resolve any problems.

Frequently Asked Questions

When can I get my results?

Please check order status [link] before calling about a specific order. Orders received by noon Mon-Thurs are usually available by afternoon the following day. Orders received by noon on Friday are usually available over the weekend, but may not be available until Mon morning.

How can I avoid delays (and make the DNACore happy)?

Orders that require special attention will be processed at the end of the daily queue and may be bumped to the next day. Special attention is often needed because of the following:

  • Tubes are submitted loose or out of order (Requires manual re-racking, which if combined with poor handwriting can make this process very difficult or even impossible. (see Why is my order on hold?)
  • Tube labels are difficult to read
  • Tube labels on side of tube (Should be on top.)
  • Samples in non-standard tubes: strip tubes, 0.2ml, 0.5ml, 2ml, screw-cap, etc. (1.5ml flip-cap tubes only please (unless submitted by air-mail))
  • Samples in non-standard plate: very flexible plate with permanent heat-seal cover or cover designed for robotic piercing (too difficult to remove without damaging plate, spilling, etc.)
  • Jumbo orders (over ~1000 samples) may be split over several days to accommodate everyday users.

Why is my order on hold?

We will make every effort to sort out order problems at our end, but we may not be able to process your order if:

  • We cannot read the labels on your tubes.
  • We cannot match your tubes to your order form.
  • The order has not been confirmed.
  • No order form was provided with your samples.
  • Tubes or plate damaged/opened and samples evaporated/mixed/contaminated.
  • Your order was mishandled at our end, but you did not supply enough template+primer for a rerun.

We will attempt to contact you by phone and/or email if there is a problem. It is helpful to have a phone number that you will actually answer. We will hold problem orders until the end of the following week before disposal.

What is your weekend / holiday schedule?

The Sequencing group operates at a reduced capacity over the weekend and is closed for MGH holidays. Samples received on Friday before noon will most likely be finished by Sat. afternoon, but not later than Monday afternoon. The courier service does not operate on weekends or MGH holidays.

Drop-Off Locations / Hours of Operation

Samples dropped off by 10 am at the following locations (note details) will be picked up Mon-Fri and delivered to our facility in time for the day's sample queue. Where provided, please use boxes for organizing tubes, and attach barcoded submission sheet with rubber band.

  • SimchesBuilding(Molecular Biology): on the 7th floor in the central corridor just outside the doors of the elevator lobby. Please note: Boxes must be put inside the cooler in the Sequencing refrigerator or else the samples will not be picked up. Card key access is required.
  • JacksonBuilding, MGH main campus: 13th floor, room 1302
  • MGHCharlestown(CNY): Room 6140 inside of room 6133, 6th floor. East side. You can contact Mathew Goodwin at 617-643-3647, with questions. Please note: Boxes must be put inside the cooler in the Sequencing refrigerator or else the samples will not be picked up.

You may also drop off your samples directly at our facility location Mon-Fri., 8am - 5 pm.

  • DNA Core facility,38 Sidney Street, Suite 100,Cambridge: on the first floor throughDNA Coredrop slot to the left of the door toSuite100. Drop box is not refrigerated, but we will check it very frequently. Building is open Mon-Fri, 8am-5pm. Please do not ring bell unless a consultation is required.

Shipping instructions

If you prefer to mail or ship your samples, please be sure they are adequately protected from crushing and from low pressure if shipped by air. We recommend high quality 1.5ml tubes or plates sealed with appropriate strip caps or very high quality adhesive seals. Parafilm has proven not to be very helpful in preventing sample evaporation from tubes or plates.

Our shipping address:

MGH DNA Core Facility
38 Sidney Street
Suite 100
Cambridge,MA02139
USA

PCR Primer Design Guidelines

Taken from

PCR (Polymerase Chain Reaction)

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.

PCR involves the following three steps:denaturation, annealing and extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice oftemperaturesis 94oC, 60oC and 70oC respectively. Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.

1. Primer Length:It is generally accepted that the optimal length of PCR primers is 18-22bp. This length is long enough for adequate specificity, and short enough for primers to bind easily to the template at the annealing temperature.

2. Primer Melting Temperature:Primer Melting Temperature (Tm) by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58oCgenerally produce the best results. Primers with melting temperatures above 65oC have a tendency for secondary annealing. The GC content of the sequence gives a fair indication of the primer Tm. All our products calculate it using the nearest neighbor thermodynamic theory, accepted as a much superior method for estimating it, which is considered the most recent and best available.

Formula for primer Tmcalculation:

Melting TemperatureTm(oK)={ΔH/ ΔS + Rln(C)}, Or Melting Temperature Tm(oC) = {ΔH/ ΔS + Rln(C)} - 273.15 where

ΔH (kcal/mole) :H is the Enthalpy. Enthalpy is the amount of heat energy possessed by substances. ΔH is the change in Enthalpy. In the above formula the ΔH is obtained by adding up all thedi-nucleotidepairsenthalpy values of each nearest neighbor base pair.

ΔS (kcal/mole) :S is the amount of disorder a system exhibits is called entropy. ΔSischange in Entropy. Here it is obtained by adding up all thedi-nucleotidepairsentropy values of each nearest neighbor base pair. An additional salt correction is added as the Nearest Neighbor parameters were obtained from DNA melting studies conducted in 1M Na+ buffer and this is the default condition used for all calculations.

ΔS (salt correction) = ΔS (1MNaCl)+0.368 x N xln([Na+])

Where
N is the number of nucleotide pairs in the primer( primerlength -1).
[Na+] is salt equivalent inmM.

[Na+] calculation:

[Na+] =Monovalention concentration +4xfree Mg2+.

3. Primer annealingtemperature :The primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Tawill produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Tamay possibly lead to non-specific products caused by a high number of base pair mismatches,.Mismatch tolerance is found to have the strongest influence on PCR specificity.

Ta= 0.3 xTm(primer) + 0.7 Tm(product) – 14.9

where,
Tm(primer) = Melting Temperature of the primers

Tm(product) = Melting temperature of the product

4. GCContent :The GC content (the number of G's and C's in the primer as a percentage of the total bases) of primer should be 40-60%.

5. GCClamp :The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

6. Primer SecondaryStructures :Presence of the primer secondary structures produced by intermolecular orintramolecularinteractions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction.

i)Hairpins :It is formed byintramolecularinteraction within the primer and should be avoided. Optimally a 3' end hairpin with a ΔG of -2 kcal/mol and an internal hairpin with a ΔG of -3 kcal/molistolerated generally.

ΔGdefinition :The Gibbs Free Energy G is the measure of the amount of work that can be extracted from a process operating at a constant pressure. It is the measure of the spontaneity of the reaction. The stability of hairpin is commonly represented by its ΔG value, the energy required to break the secondary structure. Larger negative value for ΔG indicates stable, undesirable hairpins. Presence of hairpins at the 3' end most adversely affects the reaction.