Sample Preparation

Samples can be provided as Bacteria Colonies, Glycerol Stocks, Pelleted Cultures, PCR Amplicons, or Purified DNA.  For high throughput sequencing we recommend sending the samples as pelleted cultures.  Removing the supernatant decreases the chance for contamination, reduces the weight and in turn the shipping costs.

Shipping

Samples can be sent through any carrier you choose.  It is important to note that mail is NOT delivered to our facility on Saturday or Sunday.  If you are sending samples overnight we recommend that you send them by Wednesday to provide a cushion in case of a shipping error.  If you are sending it via regular post we recommend you send them on Monday or Tuesday to ensure arrival before the weekend.

If you are located in the Madison area, please contact us to discuss pick-up options. 

Before sending samples it is important that you fill out the sample submission form.  This form should be emailed or faxed before sending your samples and a copy should be included with the shipment.  If you want to provide specific names for your samples please fill out and email us the following spreadsheet or text file. 

 

Three Important points to remember regarding all samples:

1.  We do not backup or store any samples.

     

2.  Packages are not delivered to our facilities on the weekend.  Therefore, it is vital to make sure all time sensitive packages are sent early in the week. 

3.  Contamination and poorly purified DNA are the two causes of poor sequencing results.   

 

Ways to provide samples:

Pelleted Cultures

1.  Grow colonies using Teriffic Broth in "Square Section" deep well blocks. 

•      LB does not usually provided enough growth

2.  Seal the blocks with airpore tape and shake blocks at 800 RPMs overnight, but for no

      more than 16 hours.   

•      If you can not shake your blocks at 800 RPMs we recommend shaking as vigorously as possible.  (after growth, blocks should be stored in the refrigerator, if stored in the freezer the cells may lyse)

3.  If you would like a backup of your plate we recommend removing 10ul from each well.  . 

4.  Spin the blocks at 1500 x g for 15 minutes. 

5.  Dump off the supernatant and pound on a paper towel to make sure all the supernatant 

      is removed.  (the pelleted cultures should be stored at -80 ° C until shipped) 

6.  Before shipping make sure to seal with foil tape. 

7.  Blocks should be sent on dry ice. 

Bacteria Colonies

It is important that the colonies are not too dense.  Dense populations increase the risk of contamination in the picking process. 

Bacteria colonies should be shipped on wet ice. To reduce the chance of contamination, make sure there is no condensation on the lids before securely sealing them.

Glycerol Stocks

See the recommended protocol above for growing blocks.  Follow steps one through three and then add the appropriate amount of glycerol to the bacteria stock. 

Glycerol stocks should be sent on dry ice.  Make sure plates are properly sealed with foil tape to prevent  any contamination.   

PCR Amplicons

The quality of the sequencing reaction depends on the cleanliness of the PCR product. In our lab we use the EXO/SAP protocol to clean the amplicon.  One potential problem with PCR Amplicons is that the PCR primer attachments may become damaged and therefore reduce the quality of the sequencing reaction.  To avoid this problem, we recommend that internal primers are used for sequencing because it lowers the risk of poor sequencing results due to the PCR primer attachment being damaged. 

Samples should be provided at a concentration of 50ng/ul.  The samples should be diluted using a minimum of 10ul nuclease free water*. If sending in plates for high throughput sequencing we recommend sealing the plate tightly and sending overnight on dry ice. Individual samples in separate tubes can be sent via regular post.  We recommend shipping the tube(s) in bubble wrap lined envelopes. 

Purified Plasmids

The quality of the sequencing reaction depends on the quality of the purified DNA.  We recommend using either the Eppendorf ® Perfectprep® Plasmid 96 Vac Direct Bind Kit or the Wizard® MagneSil® Plasmid Purification System.  If either of these purification methods are used  20ul of ending solution should be provided. If other kits are used we need 10ul of solution at a concentration of 100ng/ul. 

We recommend that purified DNA be sent overnight on dry ice.  It is important to make sure that the plate is sealed well to prevent any contamination in shipping. Individual samples in separate tubes can be sent via regular post.  We recommend shipping the tube(s) in bubble wrap lined envelopes. 

 

*Although we prefer you use nuclease free water you can also use a weakly buffered solution containing no more than 0.1 mM EDTA.  A suitable buffer is 10mM Tris-HCl (pH 8.5), 0.1mM EDTA.  This concentration of EDTA is lower than in typical TE buffers; excess EDTA in the template resuspension buffer can inhibit sequencing reactions.