YSBL Sequencing Submission information
All samples should be submitted using the following guidelines:
There are four ways to submit your samples:
A – Submit primers and template separately
B – Submit your primers and template premixed (if you use your own primers)
C - Submit Run Only samples
D – Submit your samples in a 96 well plate (60+ samples only)
DNA Quantity:
Template
PCR product:
100-200 bp
200-500 bp
500-1000 bp
1000-2000 bp
>2000 bp
Double stranded DNA
(plasmids)
Single stranded
Cosmid, BAC
Bacterial genomic DNA
Sample concentration
(per reaction) if using
Method A to submit.
Final amount per
reaction if using
Method B premix to
submit.
1-2 ng/µl
3-5 ng/µl
5-10 ng/µl
10-20 ng/µl
15-25 ng/µl
1-4 ng
6-10 ng
10-20 ng
20-40 ng
30-50 ng
100-150 ng/µl
200-300 ng
15-25 ng/µl
0.1-0.5 µg/µl
30-50 ng
0.2-1 µg
2-3 µg
A: Separate primers and template
1. Volume:
Samples should be in 0.5 ml tubes in 3-5 µl volumes. (Note: 2ul of sample
will be used per reaction so allow for this, plus some extra if possible, if
using multiple primers on one sample). All samples will be discarded once a
satisfactory result has been achieved, unless notified.
2. Primers: (See list on sequencing webpage)
Unless you are using the Genomic Lab primers please submit them as 3.2
µM in 5 µl volume (note: if providing your own primer please allow 1ul per
reaction.).
B: Premixed primers and template
Reaction to be set up as follows:
DNA template
Primer (3.2uM)
Water
TOTAL
x l (final conc in accordance with the table above)
1 l
x l
7.2 l
The enzyme and buffer are added by the Genomics Lab to take the final
volume to 10l.
Submit ONLY in 0.2ml domed capped PCR tubes (Axygen # PCR02D-C available in Biology Stores).
e.g., PCR product 400 bp long = 4 ng/l (need between 3-10 ng/l)
DNA template = 2 l
Primer (3.2uM) = 1 l
Water = 4.2 l
C: Run Only Samples
This only applies if you have done the sequencing reaction and clean-up
yourself using the v1.1 or v3.1 Sequencing Cycle Kit from Applied
Biosystems.
Note on the order form which Sequencing Cycle Kit you used. i.e. v1.1 or
3.1.
Submit your sample as a dried pellet.
As these can be run on the ABI3130 immediately please contact Naveed
Aziz, Claire Gay or Celina Whalley to check whether they can be
processed between the two sequencing runs of the week.
D: 96 well plate submission
Please come and see us if you have more than 60 samples to run at one
time. We can provide you with an ABI 96 well plate in which to submit
your sample, or sample + primer.
Final DNA concentration, per reaction, should be in accordance with the
table above, and primers should be 3.2 µM.
Location for all samples:
Please leave samples in the provided tray, Fridge 1 in the Genomics Lab, K241.
Order form (see sequencing website):
a. Each sample should have a unique name with no “spaces”. You can use letters and
numbers and the following punctuation only: - _
b. Download the order form complete and “Save As” YOUR NAME AND DATE .
Email it to biol-genomics@york.ac.uk
c – Please add YSBL after your name, or grant code.
Data Retrieval:
You will be notified by email when your results are ready and the data will be attached.
Run Times
Samples, and order forms, need to be submitted by midday Mondays and Thursdays with
a turn around time of 2-3 days (depending on how busy the sequencing service is!).
Any queries please contact us at the above email.