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Template
DNAs
One
of the most important factors affecting the results obtained
is the quality of the DNA template. The following is a list
of those procedures we recommend.
Plasmid DNAs
1.The
Qiagen Qiaprep Spin Miniprep Kit
2. ABI's
Alkaline-lysis/PEG prep (User Bulletin 18 from ABI)
PCR Products
1. Qiagen
Qiaquick PCR Purification Kit
2. Amicon's
Microcon 100 Columns
Gel Purified
DNAs
1. Qiagen
Qiaquick Gel Extraction Kit
2. Sigma's Agarase (click here for protocol)
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Primers
The
UM DNA Sequencing Facility supplies 18 stock primers at
no additional charge. Please indicate on your order if you
would like to use one or more of the stock primer listed
below.
M13 Universal Primer(-20) Forward
5'-GTAAAACGACGGCCAGT-3' |
M13 Universal Primer (-40) Forward
5'-GTTTTCCCAGTCACGAC-3' |
M13 Reverse Primer (-24)
5'-GGAAACAGCTATGACCATG-3' |
M13 Reverse Primer (-48)
5'-AGCGGATAACAATTTCACACAGGA-3' |
SP6 Sequencing Primer
5'-GATTTAGGTGACACTATAG-3' |
T3 Sequencing Primer
5'-AAATTAACCCTCACTAAAGG-3' |
T7 Sequencing Primer
5'-TAATACGACTCACTATAGGG-3' |
BGH Reverse Primer
5'-TAGAAGGCACAGTCGAGG-3' |
T7 Terminator Primer
5'-GCTAGTTATTGCTCAGCGGT-3' |
pQE30 Forward Primer
5'-GGAGAAATTAACTATGAGAGG-3' |
pQE30 Reverse Primer
5'-GTTCTGAGGTCATTACTGG-3' |
pBAD24F Primer
5'-ATGCCATAGCATTTTTATCC-3' |
pBAD24R Primer
5'-GATTTAATCTGTATCAGG-3' |
pGEX5 Primer
5'-GG 5'-GCTGGCAAGCCAGGTTTGGTG-3' |
pGEX3 Primer
3'-CC 5'-CCGGGAGCTGCATGTGTCAGAGG-3' |
pCMV-SP Primer
5'-GATCCGGTACTAGAGGAACTGAAAAAC-3' |
EGFPF Primer
5'-GTAGGCGTGTACGGTGG-3' |
EGFPR Primer
5'-CGTCCAGCTCGACCAG-3' |
Poly T Primer
5'-TTTTTTTTTTTTTTTTTTTTTTTTV-3' |
Poly A Primer
5'- AAAAAAAAAAAAAAAAAAAAAAAAB -3' |
MalE Primer
5'-CAGACTGTCGATGAAGCC-3' |
S-Tag Primer
5'-GAACGCCAGCACATGGAC-3' |
If you are sending
specific primers along with your template DNAs please be sure
and supply them in either 10mM Tris-HCl pH 8.0 or in water.
The primers should have a melting temperature of at least
50oC to work well in the cycle sequencing reactions. Primers
should be suppled at a concentration of 5 uM.
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Concentrations
The
following concentrations are recommended for template DNAs
and user supplied primers. Please note that that DNAs should
be supplied in either 10 mM Tris-HCl pH 8.0 or in water but
not in TE.
| Template
DNAs |
Concentration
|
Quantity
per reaction |
| Single-stranded
DNA |
10
ng/ul |
50-100
ng |
| Plasmid
DNA |
100
ng/ul |
200-500 ng |
| PCR
Fragments |
5-10
ng/ul |
up
to 100 ng |
| Large
DNA (lambda, BACs, PACs etc.) |
200
ng/ul |
1 ug |
| Bacterial Genomic
DNA |
500ng/ul |
2-3 ug |
|
Note: If attempting
to sequence very large double-stranded template DNAs the concentration
should be at least 100 ng/ul and 1 ug will be used per reaction.
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Submission
of Samples
All
DNAs, template or primer, should be supplied in 10 mM Tris-HCl,
pH 8.0 or in water but not in TE. All tubes should be clearly
labeled, sealed securely and must be accompanied by a completed
order form. Samples should be shipped either at room temperature
by overnight mail or on ice. They may also be shipped lyophilized
and we will hydrate them when they are used. If samples are
shipped by overnight letter please be sure and protect the
tubes by padding them or putting them in an additional container
to prevent breakage.
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