DNA Sequencing sample preparation

Overview

STEP ONE

Array your templates and primers in 96-well plates for large orders only. For small orders cut the 96-well plate to fit your number of samples. PCR strip tubes may also be used

STEP TWO

Seal plates with foil seals provided at drop-off sites

STEP THREE

Be sure seals adhere well so samples don't get lost

STEP FOUR

Seal plates with foil seals provided at drop-off sites. Please centrifuge the samples before dropping off.

Step-by-Step Sample Prep

How to Array and Number Your Samples
Template Concentrations & Primer Concentrations
Sequencing Primers provided by the Core

Sample Submission
Step One	Prepare Your Template DNA	Template DNA should be prepared as high-quality plasmid DNA or purified PCR product DNA. (Alternatively, you can submit plasmids as glycerol stocks and we will prep the DNA for you in advance of sequencing; please contact Li Chan for more information.
Step Two	Array Your Samples	Samples should be arrayed on non-skirted, 96-well plates and sealed with aluminum adhesive seals. Plates and seals can be found at drop-off locations. Samples should be organized as shown in the figure below (1 to 96 numbering). Please be sure to label the plate (rather than the foil seal). Please note - you can cut the plate if you're submitting a partial plate of samples and use the rest of the plate another time. We would like to avoid wasting a whole 96-well plate for, say, just 8 samples.
Step Three	Array Your Primers	Please submit your primers on a SEPARATE PLATE, arrayed in the same format as your DNA templates (so that we can simply pipet one into the other with a multi-channel pipet or robotic pipet system). Please be sure to label the plate (rather than the foil seal). Standard primers (table below) are provided by the Core at no additional charge -- please indicate on your order form if you would like us to add primer at the facility. Again, please cut the plate and save the remainder to use at another time if you're submitting just a few samples.
Step Four	Place Your Order	Place your order on-line. Please be sure to add any comments you may have regarding your samples such as High GC content, Please add primers at facility, etc.
Step Five	Submit Samples	Submit your samples (templates & primers arrayed on separate plates) by placing them in a drop-off location freezer or sending them to the facility. Alternatively, contact Li Chan to arrange pick-up from MGH, Harvard Square, or another Boston-area location.

How to Array Samples

Please number your samples as shown in white (top to bottom, left to right, one to ninety-six):

	1	2	3	4	5	6	7	8	9	10	11	12
A	1	9	17	25	33	41	49	57	65	73	81	89
B	2	10	18	26	34	42	50	58	66	74	82	90
C	3	11	19	27	35	43	51	59	67	75	83	91
D	4	12	20	28	36	44	52	60	68	76	84	92
E	5	13	21	29	37	45	53	61	69	77	85	93
F	6	14	22	30	38	46	54	62	70	78	86	94
G	7	15	23	31	39	47	55	63	71	79	87	95
H	8	16	24	32	40	48	56	64	72	80	88	96

Tips for arraying your samples:

If you have fewer than 96 samples, please array and number them by vertical column (not by row). For example, 15 samples should take up column one (#1-8) and part of column two (#9-15) with a blank noted at position 16.
If you have only a few columns of samples, please feel free to use 8-sample strips and lids instead of the plates and seals (both will be provided at drop-off locations).
Users are only charged for the samples submitted. The Core no longer charges in columns of eight.

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Template and Primer Concentrations

Please determine the concentration of your purified DNA carefully: more than 75% of sequence failures can be attributed to incorrect concentrations of template DNA.

For Plasmid DNA
Template Concentration	Volume	Primer Concentration	Volume
100 ng/µL	12 µL per well on the Template Plate	1.6 pmol/µL	12 µL per well on the Primer Plate

For purified PCR products
Template Concentration	Volume	Primer Concentration	Volume
5 ng/µL per 100 bp	12 µL per well on the Template Plate	0.4 pmol/µL	12 µL per well on the Primer Plate

For example, a concentration of 25ng/µL is appropriate for a 500bp PCR product.

Sequencing Primers

We supply the following primers (no additional charge):

T7	5'-AATACGACTCACTATAG-3'
T7 Terminator	5'-TATGCTAGTTATTGCTCAG-3'
SP6	5'-CATACGATTTAGGTGACACTATAG-3'
M13 Forward	5'-GTAAAACGACGGCCAGT-3'
M13 Reverse	5'-CAGGAAACAGCTATGACC-3'
T3	5'-AAATTAACCCTCACTAAAGG-3'

Please indicate on your order form if you would like us to add a primer at the facility.

	1	2	3	4	5	6	7	8	9	10	11	12
A	1	9	17	25	33	41	49	57	65	73	81	89
B	2	10	18	26	34	42	50	58	66	74	82	90
C	3	11	19	27	35	43	51	59	67	75	83	91
D	4	12	20	28	36	44	52	60	68	76	84	92
E	5	13	21	29	37	45	53	61	69	77	85	93
F	6	14	22	30	38	46	54	62	70	78	86	94
G	7	15	23	31	39	47	55	63	71	79	87	95
H	8	16	24	32	40	48	56	64	72	80	88	96

	1	2	3	4	5	6	7	8	9	10	11	12
A	1	9	17	25	33	41	49	57	65	73	81	89
B	2	10	18	26	34	42	50	58	66	74	82	90
C	3	11	19	27	35	43	51	59	67	75	83	91
D	4	12	20	28	36	44	52	60	68	76	84	92
E	5	13	21	29	37	45	53	61	69	77	85	93
F	6	14	22	30	38	46	54	62	70	78	86	94
G	7	15	23	31	39	47	55	63	71	79	87	95
H	8	16	24	32	40	48	56	64	72	80	88	96