+ Alkaline lysis with PEG precipitation (Protocol available upon request)
+ Qiawell
+ Amicon
+ Qiaquick (PCR products)
+ Centricon-100 Micro-concentrator columns from P-Elmer (PCR products)
Not Recommended:
+ Classic alkaline lysis
+ Boiling mini-preps
+ CsCl banding
+ Commercial glass bead/silica methods
+ Promega
Host Strain Variability in Template Preparation:
+ HB101 and DH5x host strains consistently produce good results.
+ MV1190 and XL1 Blue host strains show some variability in result quality.
+ JM101 generally does not work well.
Cleaning a failed template preparation: One of the following methods may be used to clean up a failed template preparation:
Purify the DNA by ultra-filtration (for example, with Centricon®-100 Micro-Concentrator
columns, P/N N930-2119)
Extract the DNA twice with 1 volume of chloroform or chloroform:ISO-amyl alcohol in a 24:1
(vol:vol) ratio. Precipitate the aqueous layer to remove all traces of chloroform.
Precipitate with PEG. Add 0.16 volumes of 5M NaCl and 1 total volume of 13% PEG. Incubate
on ice for 20 minutes, then centrifuge at 4°C for 20 minutes. Rinse with 70% ethanol.
DNA Quantity: The amount of DNA template used in sequencing reactions can affect data. It is important that
the concentration is accurate. Too little DNA will greatly reduce accuracy and read length. We strongly recommend gel quantification, followed by a purity check (OD 260/280)
Common problem: Not enough template.
Template Quantity
|
Template |
Quantity |
|
PCR
Product |
50-150
ng template
+ 3.2 pmoles primer in 12 µl sterile H2O |
|
Plasmid
(<9kb) |
600-800 ng
template
+ 3.2 pmoles primer in 12 µl sterile H2O |
|
Plasmid
(>9kb) |
1.0 ug
template
+ 25 pmoles primer in 12 µl sterile H2O |
|
Single-stranded |
200 ng
template
+ 3.2 pmoles primer in 12 µl sterile H2O |
Since we will be setting up
20
µl reactions, all DNA + primer should be in a
total volume of 10
µl sterile H2O. Never use TE; excess EDTA will result in poor sequencing results.
Data Format: Email; printed color chromatogram ($1.00) or Mac formatted disk (provided by user). We recommend saving the files on a disk, but only 7 chromatograms will fit on a floppy.
Results will be placed in the folder on the freezer for pick-up, after 11 a.m.
Download edit software from
Mac:
http://www.appliedbosystems.com/support/software
PC:
http://www.technelysium.com.au/chrimas14s.html
http://marketing.appliedbiosystems.com/mk/get/sss_login
(highly recommended for PC users!)
to edit and print chromatograms.
Primer Design: Usually a good sequence run will yield 600-650 accurate bases and an additional 100 bases of
less accurate sequence. Always proofread your chromatogram before ordering new primers.
If you wish to design a new primer from automated sequence data we recommend designing it
before the 550th base so you'll have 100 bases of overlap with accurate sequence.
Here are the guidelines:
-
Avoid primers with long runs of a single base
-
Primers should be between 18 & 30 bases with a Tm between 50 & 65
-
Avoid primers with secondary structure or that form dimers
-
Polystyrene 40-nanomole columns are the best choice for primer synthesis
-
If primer purification is necessary, OPC columns are a convenient option
Most Common Problems
-
Not enough template. Please gel quantify and then do an OD reading.
-
Incorrect primer dilutions, 3.2pmol is needed.
-
Promega produces
inconsistent results.
-
G/C rich templates may need a higher annealing temp. Please record Tm.
-
Two clones (templates) in one reaction.
