Template Preparation Guidelines

1) Host strains
These differ in growth characteristics, plasmid copy number, endonuclease expression and other contaminants such as carbohydrates and glycoproteins that can inhibit sequencing polymerase expression.
DH5-alpha, HB101, XL-1 Blue are consistently good. JM109 and MV1190 are usually fine but JM101 is often poor.
The growth media you use can also affect yields, LB is usually fine.

2) Template preparation
DNA that is to be sequenced must be free from other template, eg, mixed clones or PCR products, genomic DNA and RNA. Also anything that may inhibit the Taq should also be avoided eg EDTA and salt.

Plasmid Preparation
Alkaline lysis with RNAse and PEG precipitation can give very good results
There are many commercial kits available
These are usually either Ion-exchange resin based or silicon based.
Ion-exchange columns usually give very good results but care must be taken not to overload the columns. The DNA is eluted in high salt so you should always perform a desalting step, for example, a precipitation with 70%EtOH wash or use a spin column.
Silica based kits are usually cheap but can also give good results but care must be taken to achieve the best results. When the DNA is eluted in water salt is often eluted as well so it is advisable to add an extra desalting step.
Care also must be taken to avoid any silica fines in the eluted DNA (the silica fines can bleed through the columns and spell death to enzymes!) This can be done by performing a long spin on the eluted DNA and removing (and keeping!) the supernatant.

Cosmids, BACs/P1's
Alkaline lysis with PEG precipitation give good results.

PCR Products
The amount of clean-up required depends on how optimized the PCR is.
The simplest form of clean-up is simply to dilute an aliquot of the PCR reaction between 1:5 and 1:10 in water. You must insure, however that the final concentration of the PCR primers is less than 0.2uM and the dNTP's is less than 100uM. This method can give good results on highly optimized PCR's
Another method is using Exonuclease I/ Shrimp Alkaline Phosphatase. The exonuclease degrades the left over PCR primers and the alkaline phosphatase dephosphorylates the dNTP's so the won't interfere with the sequencing reaction (they tend to out-compete the flourecently labeled dNTP's). However this method does not remove any secondary products of the PCR which can lower the quality of the data.
In order to get rid of small DNA fragments column purification or gel purification is required
Column Purification: These can be silica based, gel filtration or ultrafiltration and will isolate DNA above a particular size (eg 120bp).
Gel Purification: This method is best for non-optimised reactions as it isolates the fragment you want from secondary products, primers and nucleotides. However, this method can be time consuming and the visualisation of the gel with UV light can cause "nicking" of the DNA.

Template Quantitation
Sequencers are able to handle a wide range of DNA concentrations however with very low amounts of DNA the data quality will be significantly affected.
Using UV absorbence to quantitate dilute DNA solutions tends to give widely inaccurate results.
A good way to quantitate DNA is to run an aliquot on a minigel and compare the intensity to a control of known concentration. There are also concentration ladders that are commercially available.

Material Requirements:

We guarantee to keep your templates and primers for 3 weeks. If you would like any of your materials kept for longer, you must let us know and we will move them into longer term storage. Materials in long term storage that have not been used for a period of 3 months will be disposed of unless you instruct us otherwise.

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