SHC Analytical Core Facility

DNA Sequence Request FoRM

Name____________________                                         

 

Project Number____________

 

Today's Date______________

 

 

 

Please submit your sample as follows in a  .6mL microcentrifuge tube:

Plasmid DNA:     300-500ng   (if >7kb add 100ng/kb)

          or

PCR product:        5ng/100bp

Primer:                    3.2pmol

H2O:                        to 10ml total volume

 

 

Sample Name

primer

template size (kb)

 template  

 amt (ng)

P

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IMPORTANT:

1. The computer accepts only the following special characters for sample names: -_(){}#.+  Do not use spaces. Also, if you are

    using Alphie, do not use the number symbol, #, as the program will not recognize your file names.

2. Check the P column if you want a printout of the chromatogram.

3. Indicate anything unusual about your sample in the comment column such as additives (DMSO, Betaine), annealing temperature if

    different than 50 C, etc.

4. Please highlight brightly in the comment column if you want me to add the primer.

 

SCHEDULE:

Samples submitted by 6:00pm will be cycled overnight (except Friday) to be run the following day. Data will be posted on the computer the day after the run. Files are available on Mistie Server. Go to: RESEARCH/Mistie- Research DC/ABI/PI Lab folder/file name to retrieve your data. If you need help importing files into Vector NTI, please ask for assistance.

 

 

 

 

For best sequencing results:

 

·        Resuspend/dilute template DNA and primer preferably in pure H2O only. Minimal amounts of Tris can be tolerated, but significant inhibition/interference will occur in the presence of  >0.1mM EDTA (e.g., >0.1X TE), >50mM NaCl, or small amounts of ethanol or other organic solvents.  Be sure to wash and dry precipitated DNA pellets thoroughly before resuspending!  Large pellets may require extra care to remove residual salt. 

 

·        Purify and quantitate template DNA carefully.  These are the most critical variables affecting sequence quality!  Avoid mixed or dirty templates.  If you can’t visualize a single, clean gel band, you probably won’t get good sequence.  Simple alkaline lysis minipreps are generally not clean enough for high-quality sequence data.  In our facility, Qiagen column minipreps have consistently given good results, although other methods have also been successful.  Some templates or hosts may demand particularly stringent prep quality.  Stay within the recommended range for template amounts.  The one exception may be very large templates; for plasmids >10kb you may need to increase template amount slightly.  Please work with me to optimize for this situation.

 

·        Be especially careful to purify PCR’d templates away from residual PCR reaction components and any secondary or artifact amplification products.  Gel purification, followed by extraction of the DNA from the agarose (e.g., by Qiaex kit or other silica-binding method) is highly recommended.  Even minor secondary amplification products not visible on an analytical gel may interfere with clean sequence.  When possible, use a nested primer to sequence PCR’d templates. 

 

                                                                                                           

·        Sequencing primers available from Core Facility:

      (-21)M13 Forward:            TGT AAA ACG ACG GCC AGT     

       M13 Reverse:                    CAG GAA  ACA GCT ATG ACC

       T7:                                    TAA TAC GAC TCA CTA TAG GG

       T3:                                    ATT AAC CCT CAC TAA AGG GA

       SP6:                                  ATT TAG GTG ACA CTA TAG

      Some vectors have altered these sites slightly but have retained the same nomenclature—be     

      sure to check your vector sequence!

 

·        Select custom primer sequences carefully.  Your primer Tm should be in the range of 50-60°C, since the standard sequencing labeling reaction uses a primer annealing temperature of 50°C.  Primers with Tm’s in the range 60-70°C may give better results when the labeling reaction is performed, by request, at 60°C.  Primers with Tm <50°C or >70°C are not recommended.  Tm calculation information and calculation program can be found at http://www.promega.com/biomath/calc11.htm.  In general, selection of good primers for sequencing should be guided by the same considerations used to select good PCR primers: avoid sequences with significant secondary structure or competing annealing sites within your template; aim for an evenly distributed, balanced GC content; and avoid extended runs of the same base.  If you are designing a new primer based on previously generated DNA sequence (“primer walking”), be sure the sequence is unambiguous and is from a region of good quality (i.e., always look at the original peaks, not just the text sequence!).