Sample Preparation

B-Mode

For each B-mode sample, check the Template Quantity Tables (located below) and bring this amount of template + primer sample solution in a 1.5mL Eppendorf tube at a volume of 20uL, diluted with MilliQ water if necessary. 

We will use 10uL of the final 20uL (template+primer), add our BigDye/Better Buffer mastermix and cycle as appropriate for your sample. We purify the fluorescent extension products and run them on our 3730xl DNA Analyzer. 

We will send you the chromatogram and text file and will edit them if requested. Be sure to edit your sequence data, especially the low signal regions at the start and end of the runs. A list of chromatogram viewer softwares can be found under our Software Viewers page.

House primers are also available for your use (table located below). If you would rather us add a house primer, please submit the correct DNA template amount in a 1.5mL Eppendorf tube at a volume of 16uL, diluted with MilliQ water if necessary. Designate in the "Comments" section of your iLab request that you would like a house primer added, and indicate which one. 

 

C-Mode

For experienced sequencers, you can give us 20uL of your template+primer sample solution in a 1.5mL Eppendorf tube. We will cycle, purify, and sequence your samples. The client edits and troubleshoots his/her own data. Please see the Template Quantity tables below for the amount of DNA and primer to add to each sample.

If you have lots of samples but don't want to cycle them yourself (D-mode), you can submit them in a plate. In this case, we need 10uL in each well containing 300ng template and 3.2pmol primer. Contact the lab for help if you need further assistance.

Further data processing and analysis is available on a fee-for-service basis. Also, any samples requiring extra reagents will be considered B-mode.

You will receive your sequence data, ready for editing. You should edit the base-calling for each run before assembly or further analysis. Especially note that the ends of the sequences usually have low signal, hence low quality, and should be truncated. A list of chromatogram viewer softwares available can be found under our Software Viewers page.

House primers are also available for your use (table located below). If you would rather us add a house primer, please submit the correct DNA template amount in a 1.5mL Eppendorf tube at a volume of 16uL, diluted with MilliQ water if necessary. Designate in the "Comments" section of your iLab request that you would like a house primer added, and indicate which one.

 

D-Mode

Clients can save money by doing the cycle sequencing reactions themselves. Please note that this does include cleaning up the labeled extension products prior to submitting them to the Core. This mode is appropriate for very experienced clients with proven sample preparation systems and high-throughput needs. D-mode is not for samples that require troubleshooting, additional reagents, special cycling, data processing or analysis. 

 

Template Quantity Tables


                                       Preparation Amounts for Double-Stranded Plasmids


 Size  of Plasmid (kb)Template Amount (ng) Primer Amount (pmol) Total Volume 
       L < 12 kb          600 ng           6.4 pmol      20 uL 
   L = 12 to 30 kb          ln(L)/3
           e.g. if L=15 kb
          ln(15)/3=902 ng 
          20 pmol      20 uL 
      L > 30 kb          ln(L)/2
          e.g. if L=33 kb
         ln(33)/2=1748 ng 
         200 pmol      20 uL 
 BACs, Cosmids, etc.            2 ug          200 pmol      20 uL 

 

 

                                                      Preparation for PCR Products


 Size (bp)Template Amount (ng) Primer (pmol) Total Volume 
 100-200               20 ng     6.4 pmol       20 uL 
 200-300               30 ng     6.4 pmol       20 uL 
 300-400               40 ng     6.4 pmol       20 uL 
 400-500               50 ng     6.4 pmol       20 uL 
   >500         0.1 * size (bp)
              = X ng 
    6.4 pmol      20 uL 




                                                                   House Primers List


                                    Primer Name                                                      Sequence (5' to 3') 
                                  M13/pUC(-21) F                                               TGT AAA ACG ACG GCC ATG 
                                    M13/pUC RP                                               CAG GAA ACA GCT ATG ACC 
                                           SP6                                              TAT TTA GGT GAC ACT ATA G 
                                            T3                                             ATT AAC CCT CAC TAA AGG GA 
                             T7 promoter (aka T7)                                             TAA TAC GAC TCA CTA TAG GG 
                                   T7 terminator                                                GCT AGT TAT TGC TCA GCG G 
                                         pGEX-5'                                        GGG CTG GCA AGC CAC TGG TGG TG 
                                         pGEX-3'                                         CCG GGA GCT GCA TGT GTC AGA GG 
                                      pQE-9F115                                            TAT GAG AGG ATC GCA TCA CCA T 
                                      pQE9-R348                                            TTG GGA TAT ATC AAC GGT GGT A