CCHMC Genomics Sequencing Facility ADMIN

SEQUENCING FROM PLASMIDS

Introduction

This service generates the sequence of a purified plasmid. You can use any primer that anneals within the plasmid to initiate the sequence. Most commercially available plasmids contain universal priming sites. The facility provides the following universal primers at no charge for your sequencing reactions:

#	Primer Name	Length	Sequence (5' to 3')	Tm
1	SP6	18	ATTTAGGTGACACTATAG	42.1
2	T7	20	TAATACGACTCACTATAGGG	47.5
3	T3	20	AATTAACCCTCACTAAAGGG	49.3
4	M13	24	CGCCAGGGTTTTCCCAGTCACGAC	64.1
5	M13(-21)	18	TGTAAAACGACGGCCAGT	54.4
6	M13R	22	TCACACAGGAAACAGCTATGAC	54.8
7	T7term	19	TATGCTAGTTATTGCTCAG	46.3
8	BGHrev	19	TAGAAGGCACAGTCGAGGC	56.8
9	GL2	23	CTTTATGTTTTTGGCGTCTTCCA	54.1
10	RV3	20	CTAGCAAAATAGGCTGTCCC	53.1
11	VP1.5	18	GGACTTTCCAAAATGTCG	48.7
12	XL39	20	ATTAGGACAAGGCTGGTGGG	57.0
13	CMV-Forward	21	CGCAAATGGGCGGTAGGCGTG	63.9
14	SV40pA-R	18	GAAATTTGTGATGCTATTGC	47.9

The plasmid must be purified from a cleared bacterial lysate without residual proteins, salts, phenol, or ethanol contaminants and must be resuspended in a buffer free of EDTA. This can be done using one of several methods:

Silica matrix capture column (like Qiagen's QIAquick)
Phenol/Choloform extraction followed by ethanol precipitation
Silica coated magnetic beads purification (like Promega's Magnesil)

Sample preparation

For each sequence, prepare the template (purified plasmid) and the sequencing primer mixed together in a single tube (or single well of an 8-tube strip or 96-well plate). Please note that if multiple sequences are to be generated from the same template you must submit that template multiple times. You will need to provide 100 ng per kb of total plasmid size (an 800bp insert cloned into a 3.2kb pTOPO vector will require 400ng of plasmid to be submitted for sequencing) together with 7 pmol of the sequencing primer in a total volume of 12ul. If you want to use one of the universal primers listed above, the facility will add it for you. In this case, you would simply submit the plasmid in a total volume of 10ul.

Our automated protocol requires the use of plasticware (tubes, strips & 96-well plates) of uniform dimensions that we provide at no charge. You can find the plasticware at any of our drop-off locations near the barcoding station. The use of plasticware with improper dimensions will delay the processing of your samples.

Acceptable vial types for the robotic liquid handler are 1.5 mL centrifuge tubes, 0.2 mL 8-strip tubes with a single, removable cap and fully-skirted V-bottom 96-well microplates. Other vial types (0.5mL centrifuge tubes, 8- or 12-strip 0.2 mL vials with individual caps or non-skirted pcr plates) will not be accepted. To help ensure the proper identification of the orientation of samples within an 8-strip vial, we ask that these strips not be clipped down to fewer than the 8 original wells present in the vial.

First timer's walkthrough

Prepare your sequencing sample by mixing together in a single tube (or well) primer, template and water in a total of 12ul. Skip the primer addition step if you are using one of the facility provided universal primers.

TEMPLATE:
Prepare your plasmid from a bacterial pellet using the Qiagen Qiaquick Spin plasmid preparation kit. Quantify the amount of plasmid in your extraction by reading the OD260 of the solution and multiply by 50, this gives a concentration in ng/ul. Use the appropriate amount to make the final quantity 100ng per kb of total construct size.

WATER:
Bring template mixture to a total of 10 uL with molecular biology grade water.

PRIMER:
Make a 20uM stock of your primers. From this stock, make a 3.5uM working solution for sequencing purposes. Use 2uL (7pmol) in each sequencing reaction. If you need to convert from ng/uL concentrations to molar concentrations, you can use our Primer Concentration Calculator. The final volume for samples submitted with custom primers is 12uL.

Submission process

• Log in to our site using your CCHMC network ID and password, or StratoCore userID and password if non-CCHMC.
• Select the online submission form and choose the SANGER METHOD DNA SEQUENCING submission type.
• The prompts will guide you through the process. You will need to have the following information to fill out the electronic form: name of submission, type of plasticware in which the samples will be submitted (tubes, strips, plates), names of samples and primers and how many of each you are submitting as well as which samples will receive facility provided universal primers (if any).
• Make sure to hit the ACCEPT button at the end of the process to complete your submission.
• You will receive an email with an invoice summary for your records.
• Proceed to the delivery of samples to our drop-off location (First floor, 'R' building) and register them using the barcode scanner as indicated in the how to submit section.

Adjuvants

To assist in the sequencing of difficult to sequence templates, the CCHMC Genomics Sequencing Facility makes available three common adjuvants to incorporate into the sequencing reaction mixture. These adjuvants are Betaine, DMSO and Formamide, which are selected per sample in the online submission interface and added by our robotic liquid handler. There is no additional cost for the addition of one of these adjuvants.

Sample types that may benefit from the addition of an adjuvant to the sequencing reaction are:
• Samples with high GC content (>~57%) in the region to be sequenced
• Samples that include a structural element, such as a hairpin or other loop structure

An adjuvant must be selected at the time of submission, and cannot be added to a sample at a later time. If the sequencing quality of a sample is poor and selected to be repeated, an adjuvant cannot be added to the sample at that time. In this case, if the reaction is thought to have failed due to the necessity of an adjuvant, a new online submission must be made to request the addition of the adjuvant.

Repeated Samples

The CCHMC Genomics Sequencing Facility will repeat several low-quality, or 'failed' samples from nearly every Sanger Sequencing run for our own internal Quality Control purposes. Samples to be repeated are selected in a quasi-random fashion and no additional cost is incurred for these repeats by the requesting investigator. Seqeuncing data from the repeated sample is compared to the original and evaluated for quality. Nearly all repeated samples should produce similar quality metrics as the original sample from the original run. If you have a sample that has been repeated, you will receive an additional set of data files (.ab1 and .seq) for the repeated sample, designated as repeats by adding the suffix '_R' to the sample name in the files. For additional information about Sanger repeats, please refer to our Repeat Policy, found here.