Where can i get an E. Coli gene sequenced for cheap ?

[Hugues]

Hi guys,

I have 2 samples of E. Coli for which i would like to have a specific gene sequenced,

where is the cheapest place i can get that done ?

I checked online, there are many, not sure where to start , any recommendations ?

i live in Switzerland

thanks

[Kermit Henson]

macrogen or bgi 

[Mega [Andreas Stuermer]]

Basically any synthesis company will sell you primers ("oligos"). And they will also do a sequencing run. Make sure to inquire about prizing first. Total costs should be 10 to 20$ plus shipment of your DNA to them.

[bioscisam]

If you are in switzerland GATC is based in Konstanz and normally have reasonable prices.

Also Eurofins Genomics are based in southern germany. Normally you just post your sample to them with a barcode and get the sequence after a few days by looking at your account info.

[Hugues]

Hello,

10 to 20 usd ??

mmm....

Here is a quote i received , for 760.80 usd for 2 samples, shipping charges excluded

Are we talking about the same work here ? Sorry i'm a newbie to this.

Can you give me the name of a company doing this work for that kind of price ? I will contact them.

***************************

Our Offer

1. DNA isolation

DNA isolation (column) 2 - 160.40 320.80

Total 320.80

- DNA will be isolated from your samples using an appropriate kit. 

2. Primer design & synthesis and PCR testing and optimization

Primer design including synthesis 2- 25.00 50.00

PCR set-up and optimization 1 300.00 300.00

Total 350.00

3. PCR clean-up and Sanger sequencing

Single PCR purification, price per sample  2- 9.00  18.00

Economy Run 4- 18.00  72.00

Total 90.00

- PCR products will be purified and bi-directionally Sanger sequenced. The results of the Sanger

sequencing will be sent to the customer for further analysis.

Total costs for the characterization: CHF 760.80

*****************

[Abizar Lakdawalla]

Two kinds. If you are sequencing a gene of less than 1000 bp then you would use Sanger sequencing which is read by capillary electrophoresis. That costs about $10. If you are sequencing the whole Genome of E.coli, 5,000,000 bp, then that would cost $100's.

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[djwr...@gmail.com]

How long is the gene in base pairs? 

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[Hugues]

about 373 according to this article:

https://docs.google.com/document/d/1F0zYVcIuhWTjmzizaQ7M3UiRLq9M5GpUbP4NXhnKphU/edit

or this:

https://www.ncbi.nlm.nih.gov/nuccore/11120596?report=fasta

[hlalibe .]

i asked them to sequence only one gene, and they indeed specify SAnger sequencing in their quote.

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[djwr...@gmail.com]

Do you have a primer for it? 

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[Bryan Jones]

That quote seems very high. The 10-20 USD price wouldn't include primer design & synthesis. But even taking that into account, this is way too high. DNA isolation costs a few bucks at most for materials. 

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[Hugues]

no i don't have a primer, they need to provide

it's included in the quote i showed in my earlier post

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[djwr...@gmail.com]

Pick a primer 50 to 100 bp upstream of the gene (20 base pairs) or even better look for a common sequencing primer provided by sequencing company for free.  How are you sending the sample? Is it a colony?

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[Hugues]

i take a small sample of my colony, put in a small tube with alcohol, and ship.

[bioscisam]

Sounds very expensive for what it is.

Depending on what you currently have access to you can extract the DNA yourself and create a PCR product which you can then clean up and send off for sequencing along with the primers you used to amplify it.

If I'm amplifying something out of E.coli I tend to just do a boiling lysis (colony in a PCR tube with H2O and 10-15 mins at 95C +) then use a couple of microlitres of the supernatant of that in a PCR reaction as template.

Seems hugely overkill that they are offering a full on premium extraction just for that (sales rep bumping up the invoice maybe?)...

Design primers as Roninlaw mentions above. I tend to order mine from Eurofins Genomics / MWG (whatever they're called these days) they're based in southern germany but the DHL or FedEx things to me in England within a couple of days so fairly handy.

I normally expect spend 2 or 3 GBP per primer.

The PCR product/reaction can then be cleaned up using a kit like https://www.promega.co.uk/products/dna-purification-quantitation/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/   (that's the one I use, there is a method for this process in sambrooks/maniartis but it's more trouble than it's worth vs just paying for a kit, that one is 75GBP list price for 50 preps so that works out to about £1.50 per prep).

Then send that cleaned up reaction and some of each primer as per the sequencing company's requirements. One run of sanger sequencing tends to cost anything like 5 - 10 GBP. The pricing tends to get a bit better if you pay for several in advance.

So I reckon you could at least knock a zero off the end of the price their offering if you're equipped and willing to do some basic molecular biology. :)