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DNA SEQUENCING SERVICE
The
MMG Core Facility is now located in room MSB1.004 and provides automated
DNA sequencing services for all interested researchers within the UT Health
Science Center as well as other academic institutions.
Sample Preparation
Guidelines [click here]
Sample Submission
Form [PDF]
Obtaining Your Results [click here]
Primers We Provide [click here]
Technical Information [click here]
BIACORE SYSTEM
The
MMG Core Facility houses a Biacore 2000 instrument, which employs state-of-the-art
surface plasmon resonance technology and microfluidics for the real-time
analysis of macromolecular interactions. Contact the Core Facility
to obtain instrument training and to discuss use of the Biacore system
(713-500-5455 or core@uth.tmc.edu)
REAL TIME PCR
The
MMG Core Facility houses a ABI 7500 Real Time PCR System (96 well plate).
Real time PCR is available at $25 per run. This price includes only the
run. Reagents and consumables are not included in this price. Contact
the Core Facility for more infromation (713-500-5455 or core@uth.tmc.edu)
SEQUENCING SAMPLE PREPARATION
GUIDELINES
1. Prepare
both the primer and the template DNA in deionized water.
2. Follow link on Core homepage for a list of vector primers we can provide.
3. Dilute your custom primer to a concentration of 5 pmol/ul (5 uM).
At least 3 ul is required for each reaction. Please provide a little
extra.
4. 0.5 ug of ssDNA or 1.0 ug of dsDNA at a concentration of 200 ng/ul
is required for each reaction (except as noted below).
5. Please indicate on the submission form if the template to be sequenced
is larger than 15 kb or smaller than 1kb.
6. To sequence clones much larger than 15 kb (e.g., lambda or BAC clones),
please increase the template concentration to 500 ng/ul and provide your
custom primer at a concentration of30 pmol/ul (30 uM).
7. For PCR products and other templates that are shorter than 1.0 kb,
the amount of template needed is determined by the length. For each reaction,
25 to 50 ng/100 bp is recommended in a final volume of 10 ul.
8. There is no need to aliquot your template or primer if it is being
submitted for
multiple reactions.
SEQUENCING PRIMERS
PROVIDED
| Primer |
Sequence
(5' to 3') |
| M13 Forward (-21) |
TGT AAA ACG
ACG GCC AGT |
| M13 (-40) |
GTT TTC CCA
GTC ACG AC |
| M13 Reverse |
GGA AAC AGC
TAT GAC CAT G |
| T3 |
AAT TAA CCC
TCA CTA AAG GG |
| pBST3 |
CCT CAC TAA
AGG GAA CAA AAG C |
| T7 |
GTA ATA CGA
CTC ACT ATA GGG C |
| EXT7-1 |
TAA TAC GAC
TCA CTA TAG GGC GA |
| EXT7-2 |
TAA TAC GAC
TCA CTA TAG GGA GA |
| EXT7-3 |
TAA TAC GAC
TCA CTA TAG GGG AA |
| T7-Terminator |
GCT AGT TAT
TGC TCA GCG G |
| SP6 |
CAT ACG ATT
TAG GTG ACA CTA TAG |
| SK |
CGC TCT AGA
ACT AGT GGA TC |
| KS |
TCG AGG TCG
ACG GTA TC |
| CMV-F |
CGC AAA TGG
GCG GTA GGC GTG |
| BGH-R |
TAG AAG GCA
CAG TCG AGG |
OBTAINING YOUR SEQUENCING
RESULTS
Currently we can provide the
ABI file and a simple text file on your floppy, zip (100) disc, CD or
(USB) flash drive, and a hard copy of the electropherogram.
To open your ABI file, download Finch TV (for Linux, PC, or Mac OSX; click
here to install) Chromas (for PC; click
here to install), or EditView (for Mac OS9; click
here to install) that is compatible with your computer. All three
programs are free.
TECHNICAL SEQUENCING
INFORMATION
For DNA sequencing, we employ:
- chemistry: cycle sequencing,
dye terminator (Unidirectional PCR reaction using terminators labeled
with different fluorescent dyes. Our current standard dye is Applied
Biosystems BigDye® Terminator v.3.1)
- Post-PCR cleanup paramagnetic
beads (Agencourt)
- Separation capillary electrophoresis
(ABI 3100, ABI 3100-Avant) Maximal capacity: 120 separations/24hr
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