Preparation of amplicons for shipping to Albany

for sequencing.

 

Guideline:

Ideally, each plate should contain the following amplicons per primer: 2 of each T.urartu, Ae. tauschii, and Ae. speltoides, and at lest 8 of Langdon). Well H11 and H12 must be empty. They will be used for sequencing controls.

 

Purification of amplicons:

Only strong single band PCR products could be purified and mailed for sequencing.

 

Treatment with Exonuclease I (USB cat#: 70073Z or 70073X) and Shrimp Alkaline Phophotase (USB cat#: 70092Y, 70092Z, 70092X) to remove excess of dNTPs and primers:

 

5 ul of PCR product

2 ul of SAP (2 units)

1 ul of ExoI (10 units)

 

Incubate at 37C for 15 min, and then inactivate enzymes at 80C for 15 min.

Freeze and store at –20 C or –70C until needed.

 

After treatment with Exonuclease I and Shrimp Alkaline Phosphatase add 8 ul of deionized water to reaction, and mix by pipetting up and down. Transfer 3 ul aliquot of diluted reaction mix into semi-skirted 96-well PCR plate (GeneMate, Cat#: T-3085-1, to order call 1-800-999-2901). Add 1ul of the F primer (which is one of the primers used for PCR diluted to the concentration 3.2 pmole/ul). Prepare mix of the amplicon also with the R primer. Freeze and ship on dry ice. Thus for each plate of Exo-SAP purified amplicons you should ship two plates for sequencing: one with the F primers and another with the R primers.

 

 

Preparation of Excel spreadsheet:

 

Excel spreadsheet should accompany each sequencing plate. Use thee letter lab designator in the name of the Excel file (UCD for J.Dvorak’s lab, KSU for B. Gill’s lab etc.) followed by tree digits showing the sequencing plate number (UCD001 – the first plate send for sequencing from J. Dvorak’s lab). Every lab should keep track of the amplicons and plates mailed to Albany.

 

The names of amplicons send for sequencing should be listed in the accompanying Excel file. We suggest using the following naming scheme:

 

 

 

  1. Accession number of the mapped EST (8 symbols).
  2. Designation of the genomic DNA used as template for PCR reaction:

Tu01, Tu02 -  T.urartu DNAs

At01, At02 – Aegilops tauschii DNAs

As01, As02 – Aegilops speltoides DNAs

Ld01, Ld02 …..Ld10 – Amplicons of individual TA Langdon clones

  1. Primer plate number (P001 is WSNP001 plate received from MWG).
  2. The location of the F (left) PCR primer in the P001 plate.
  3. Primer plate number (P001 is WSNP001 plate received from MWG).
  4. The location of the R (right) PCR primer in the P001 plate.
  5. Amplicon number, starting  from the top of the gel (if gel  purification is used). If not, use default 1.
  6. Sequencing primer: F – forward (corresponds to forward PCR primer), R – reverse (corresponds to reverse PCR primer).
  7. Attempts to sequence an amplicon (1 - first attempt, 2 – second attempt).

 

See the attached Excel spreadsheet partially filled with sample names. The only fields which must be filled by the sample sender is the Plate name in A2 position of the spreadsheet and the Sample names in the B column. Then Excel file must be saved in Tab-delimited format and send to Albany as an e-mail attachment.

 

 Note: Genome-specific amplicons are generated with one conserved primer (plate and position in the plate) and
one genome-specific primer (plate and position in the plate). Therefore, two primer plate numbers are for a  conserved primer and a genome-specific primer,  For conserved primer generated amplicons, the same plate  number will be inserted to both fields since both primers are in the same plate.