Difference between revisions of "Help:2019 DNA Distribution"

Revision as of 16:27, 26 March 2019

	The 2019 DNA Distribution Kit
	Whether you’re new to iGEM and the Registry of Standard Biological Parts or an experienced participant, make sure to read through the Distribution Handbook.

The iGEM 2019 DNA Distribution contains over 2000 high-quality part samples as dried plasmid DNA. Each sample is QC tested through sequencing and AB test plates.

While there is not enough DNA for 3A Assembly right out of the kit, you can transform a part into competent cells and make your own glycerol stock for future use.

When you receive your Distribution Kit, let us know through twitter (#WelcomeToiGEM)! If you're new to the Registry, iGEM, or synthetic biology, you will also want to read through our Help pages before you get started.

What's included in the 2019 Distribution

Your 2019 DNA Distribution contains the following:
DNA Distribution Kit Plates 1 - 6
Linearized Plasmid Backbones
Competent Cell Test Kit
Measurement Kit
Where's my Sample Submission Kit?
iGEM Stickers and Pins!
Sponsor Material

If there is an issue with your distribution kit, please send an email to hq (at) igem . org

Storage Instructions

Please keep kit components out of direct sunlight for long term storage, and follow individualized temperature storage requirements below.

Freeze Indicator included in the Distribution Kits: If your kit was frozen at some point, the indicator would turn purple.

• DNA Distribution Kit Plates: Store at room temperature. When a well is resuspended, store plate at -20°C, or store resuspension separately at -20°C.
• Competent Cell Test Kit: (after resuspension) store -20°C
• Linearized Plasmid Backbones: Store at -20°C
• Measurement Kit: Store at room temperature or above 4°C. Do not store below 4°C (e.g. -20°C)

Note: If you received your kit, and the freeze indicator was purple (instead of clear), then at some point during shipment your kit was frozen. Your Measurement Kit (the silica beads specifically) is no longer usable, but you can request a new one from us.

DNA Distribution Kit Plates

The distribution only contains parts with samples that are sequence confirmed or ends confirmed (long parts > 1600bp). This has ensured a high quality distribution, in which all parts have a sequence result of Confirmed, Partially Confirmed/Single Error or Long Part. All users should review the sequence results of a part sample before using it. Note: We're still in the process of curating the Distribution, and removing samples that did not meet our requirements!

The majority of part samples in the in the Distribution Kit are in pSB1C3, the Registry’s standard shipping plasmid backbone.

Why is this useful?

• all parts are flanked by the BioBrick prefix and suffix.
• pSB1C3 has our standard primer sites (VF2 and VR), so you can sequence all parts with the same primer set.
• pSB1C3 has a high copy origin, improving yields for minipreps
• pSB1C3 is chloramphenicol resistant, so growing and maintaining parts only requires a single antibiotic

Storage: The distribution kit plates are comprised of dried DNA with cresol red dye, so they are stable at room temperature. However, once the DNA is resuspended in any of the wells, we recommend either storing the kit plate with its plastic cover in a -20°C freezer, or aspirating the rest of the resuspended DNA from the well and keeping it separately in a -20°C freezer.

Competent Cell Test Kit

Whether you’re purchasing competent cells or making your own in the lab, you should test their efficiency before you start transforming parts in the Distribution Kit. The Competent Cell Test Kit is a standardized way to test and calculate the efficiency of your competent cells. If you have issues with using the parts in the Distribution Kit, the first thing we’ll need to know is the efficiency of your competent cells.

See the Competent Cell Test Kit page for more in-depth instructions.
Storage: When resuspended the Competent Cell Test Kit should be stored at -20°C, for longer term storage.

Linearized Plasmid Backbones

The 2019 Distribution Kit includes a set of linearized plasmid backbones: pSB1A3, pSB1K3.m1, and pSB1C3. These plasmid backbones have been prepared via PCR and purified. Prior to ligation the linearized backbones will need to be digested with with EcoRI, PstI and DpnI (DpnI is optional: to cut up original template DNA used to create linearized plasmid backbone), leaving two ends ready to be ligated to a BioBrick part.

The Registry has phased out tetracycline but if you need pSB1T3 as linearized plasmid backbone, you can make your own or request some from the Registry.

See the Linearized Plasmid Backbone protocol page for more in-depth instructions on how to use them and make your own.
Storage: The Linearized Plasmid Backbones (pSB1C3/pSB1A3/pSB1K3: 25ng/ul at 50ul) should be stored at -20°C, for longer term storage

Measurement Kit

Over the past four years, iGEM has advanced the frontiers of science with the two biggest interlaboratory studies ever done in synthetic biology. These studies established a baseline for replicability of fluorescence measurements and identified likely key sources of error, and have now been published as an open-access journal article in PLOS ONE. To read the article, go to the following URL: http://dx.doi.org/10.1371/journal.pone.0150182.

Please note, that there is no InterLab activity is completely optional and is not required for any team.

This Measurement Kit is for teams to use for their own experiments.

The kit includes 10 tubes (5 tubes of Fluorescein Sodium Salt, 3 tubes of LUDOX, 1 tube of silica beads). Parts to be measured in the study are in the DNA Distribution Kit.

See the [http://2019.igem.org/Measurement/InterLab InterLab Study page]] for more in-depth instructions.
Storage: The Measurement Kit should be stored at room temperature or higher 10°C. Do not store below 4°C as this causes issues with the silica beads (ex. below 4°C, -20°C)

Part Submission Kit

All part submissions must be shipped as dried DNA, in 96 well plate format, and covered with adhesive foil. We've provided a submission kit with all of the materials you'll need to ship your parts.

The Submission Kit includes:

• (3) 96-well skirted PCR plates
• (3) universal plate lids
• (4) adhesive foils
• bubble wrap
• a box with the iGEM HQ address, for shipping your submission

See the Submission Kit page for more in-depth instructions.
Storage: The 2018 Submission Kit should be stored at room temperature

Getting Started

Locating a Part in the Distribution

Before using the DNA plates, you should search the Registry for useful parts, which will also tell you if we have sample in stock, its location (if they're in your 2018 Distribution), requirements, quality control, etc.

You can browse the contents of the 2018 Distribution in their entirety by visiting the Repository.

Searching

• The Catalog of Parts and Devices will allow you to browse parts and devices by various criteria, including function, chassis, standard, etc.
• The Search Menu will let you search by text or part name.
• It is also possible to see the contents of the Kit Plates in their entirety. You can look at the DNA Repositories section on the main page.
  • Click on 2018 Distribution to see the contents of all of your kit plates.
  • Click on the 2018 Kit Plate of your choice, which will list all parts by their part name (BBa_..) in a plate along with their quality control information. Or you can click on the small part diagram below each Kit Plate link: "See a summary of the parts in this plate."
  • These options will show you what is in each well of your plate, however they are not the best way to find specific parts you would like to use.

In stock

If you find a part that you would like to use, you need to make sure that a sample of the part is in stock. There are many parts on the Registry that people are still working on, or have decided not to continue working on anymore. This of course means that the DNA is not available in the Registry's Repository. The simplest way to tell whether the part has a sample is to look at the top right of the part's Main Page. If the part has an available sample the top part of the box will be green and say "Sample In stock."

Requesting a part

We've taken care to create a very well-rounded DNA Distribution this spring, however, should you find the part, or parts, that you require are not available in the 2018 DNA Distribution but are available elsewhere in the Registry's Repository, send us an email (hq [AT] igem [DOT] org) in order to request a sample. Include the part name, the plasmid it's located in, the source, and the quality control information, and we'll send it out to you. For more information please see the Requesting Parts page

Using the online QC resources

Here at Registry, we want everyone to take a look at the results of the quality control measures we've taken this year and previous years, in order to make an informed decision when choosing to use a part. We've made sure to update the online repository for the Spring 2018 distribution with our quality control results.

The best way to use our quality control information is to use it on a part by part basis. As you design your project, make sure to check every part that you're interested in for its QC data. After searching for a part in the registry and arriving at its main page, click on the Get This Part link which will take you to the section listing various quality control information for a samples of that part and its location within the registry.

Using the Spring 2019 DNA Distribution

Storage

DNA Kit Plate Orientation

Locating Your Part from iGEM Videos.

Top view of plates containing dry DNA; red circle indicates well 13H

The foil covers on the (384 well) Kit Plates can be easily punched through with a pipette tip. Unfortunately, the foil cover will also obscure both column and row markings. You can still find your part by correctly orienting the plate using the two notched corners as markers: well A1 is located at the upper left corner of the plate when the long side of the plate with the notched corners is considered the bottom.

Make sure that the two notched corners of the plate are oriented at the BOTTOM of the plate (see the "top view" image on the right for correct orientation)

DNA Kit Plate Instructions

Before you use the DNA in the Distribution Kit Plates, be sure to test the efficiency of your competent cells with the Competent Cell Test Kit.

To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µL

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to [http://openwetware.org/wiki/Miniprep/Kit-free_high-throughput_protocol miniprep] the DNA AND make your own glycerol stock (for further instruction on making a glycerol see [http://openwetware.org/wiki/Endy:Making_a_long_term_stock_of_bacteria this page]). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.

Note: There is not enough DNA in each well to perform anything but transformations

Get & Use...