Difference between revisions of "Help:2013 DNA Distribution"

Revision as of 20:35, 5 June 2013

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	The 2013 DNA Distribution Kit
	The DNA Distribution has been entirely revamped this year! Whether you’re new to iGEM andthe Registry of Standard Biological Parts or an experienced participant, please make sure to read through the Distribution Handbook.

The 2013 DNA Distribution contains over 1000 part samples as dried (miniprepped) DNA, with each sample QC tested through sequencing, AB test plates, and restriction digests. While there is not enough DNA for assembly, you will be able to transform the DNA into competent cells and then make your own glycerol stocks of any part you wish.

If you're new to the Registry, iGEM, or synthetic biology, you'll want to read through our Help pages before you get started. The Learn section is the best place to start, and will take you through the basics.

Changes to the 2013 DNA Distribution

We’ve made some important changes to this year’s DNA Distribution!

What's included in the 2013 Distribution

Your Spring 2013 Distribution contains the following:
DNA Distribution Kit Plates 1 - 4
DNA Distribution Kit Plate 5 (Supplemental Plate)
Linearized Plasmid Backbones
Transformation Efficiency Kit
iGEM Stickers and Pins! (iGEM teams only)

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

DNA Distribution Kit Plates

In making this year’s distribution, we selected only parts with samples that were sequence confirmed or ends confirmed (long parts > 1600bp). All parts underwent an additional round of our quality control process: sequencing, restriction digests & gels, and antibiotic testing. This has ensured a high quality distribution, in which all parts have a sequence result of Confirmed, Partially Confirmed or Long Part. However, be sure to take a look at the quality control results before using a part! Note: We're still in the process of curating the Distribution, and removing samples that did not meet our requirements!

Storage: The distribution kit plates are comprised of dried DNA, 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 -20C freezer, or aspirating the rest of the resuspended DNA from the well and keeping it separately in a -20C freezer.

Kit Plates 1 - 4

In addition to the new parts that have been submitted to the Registry, we have transferred over many of our existing sequence verified parts into pSB1C3, the Registry’s standard shipping plasmid backbone. All parts in DNA Distribution Kit Plates 1 to 4 are in pSB1C3.

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

Kit Plate 5

Kit Plate 5 is a supplemental plate for the 2013 DNA Distribution. Unlike Kit Plates 1 - 4, Kit Plate 5 does not have parts in pSB1C3.

Instead, Kit Plate 5 contains:

• high quality parts which have yet to be transferred into pSB1C3
• alternate samples for parts that already have a sample with pSB1C3.

Transformation Efficiency Kit

Whether you’re purchasing competent cells or making your own in the lab, you should test their efficiency before you use them with the parts in the DNA Distribution Kit Plates. The Transformation Efficiency 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 Transformation Efficiency Kit page for more in-depth instructions.
Storage: The Transformation Efficiency Kit should be stored at 4C or -20C.

Linearized Plasmid Backbones

The 2013 Distribution Kit includes a set of four linearized plasmid backbones: pSB1A3, pSB1C3, pSB1T3, and pSB1K3.m1. 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. All 2013 submissions will need to be in pSB1C3, so we recommend using our pSB1C3 linearized backbone for that purpose.

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 (25ng/ul at 50ul) should be stored at 4C or -20C.

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 2013 Distribution), requirements, quality control, etc.

You can also browse the contents of the 2013 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 2013 Distribution to see the contents of all of your kit plates.
  • Click on the 2013 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 2013 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 2013 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 2013 DNA Distribution

Storage

• DNA Distribution Kit Plates: The distribution kit plates are comprised of dried DNA, 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 -20C freezer, or aspirating the rest of the resuspended DNA from the well and keeping it separately in a -20C freezer.
• Transformation Efficiency Kit: The Transformation Efficiency Kit should be stored at 4C or -20C.
• Linearized Plasmid Backbones: The Linearized Plasmid Backbones (25ng/ul at 50ul) should be stored at 4C or -20C.

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 Transformation Efficiency 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/ul

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 10uL 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. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1ul 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

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