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BioLab Manual: Difference between revisions

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BioLab Manual (view source)

Revision as of 15:15, 26 April 2014

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Also see general hackspace [[suppliers]] page.
Also see general hackspace [[suppliers]] page.
=== DNA extraction===
* Triton X-100
*Triton + TBE + meat tenderiser solution.
*Proteinase K solution (We think it's 10mg/ml but not 100% sure)
*Chelex 100 sodium form. (25g) from [http://www.sigmaaldrich.com/catalog/product/SIGMA/C7901?lang=en&region=GB sigma] for £55.08. We also have some of this suspended in tap water
*70% isopropanol (rubbing alcohol) - widely and cheaply available
===PCR and restriction digest===
*[https://www.sigmaaldrich.com/catalog/product/sigma/p2893?lang=en&region=GB Taq ReadyMix]. Click on bulletin for useful info about procedure for using it. £144 + £30 shipping for 100 reactions from [http://www.sigmaaldrich.com/catalog/product/sigma/p2893?lang=en&region=GB sigma]. We can halve reaction volume with no adverse effects giving us 200 reactions.
* [http://www.sigmaaldrich.com/catalog/product/sigma/s4438?lang=en&region=GB SYBR green taq readymix] £115.92 for 200 reactions
*dH20 (deionised water bought from garages).
* [https://lists.kentgeek.org/pipermail/lhs-biohacking/2012-January/000210.html Post] on reconstituting primers
'''Amelogenin'''
Gene associated with tooth mineralisation on human X and Y chromosomes.
Sequences: 5'-CTGATGGTTGGCCTCAAGCCTGTG-3' and 5'-TAAAGAGATTCATTAACTTGACTG-3'
Produces product 977 bp long (human X chromosome) or 788 bp long (human Y chromosome).
'''Amelogenin'''
Gene associated with tooth mineralisation on human X and Y chromosomes.
Sequences: 5'-CCCTGGGCTCTGTAAAGAATAGTG-3' and 5'-ATCAGAGCTTAAACTGGGAAGCTG-3'
Produces product 106 bp long (human X chromosome) or 112 bp long (human Y chromosome).
Anneal at: 66 C (source: http://biowww.net/gene/gene-AMELY.html)
'''SRY'''
Sex-determining region Y.
Sequences: 5'-ATAAGTATCGACCTCGTCGGAAG-3' and 5'-GCACTTCGCTGCAGAGTACCGAAG-3'
Produces product 93 bp long (human Y chromosome).
* KPN1 (£27 for 1000 units from [http://www.sigmaaldrich.com/catalog/product/sigma/r1258?lang=en&region=GB sigma] - a unit is "the enzyme activity that completely cleaves 1 µg λ DNA in 1 hr. at 37 °C in a total volume of 25 µl of Buffer SL for restriction enzymes.". Quantity not listed but I estimate 115ul) and ALU1 (£18.90 for 100 units from [http://www.sigmaaldrich.com/catalog/product/sigma/r6885?lang=en&region=GB sigma] - can also buy 500 units for £33.80) for blood typing. I guess we will be using it on minimum 20ng of DNA, which gives us 50 reactions for Kpn1 and 5/25 reactions for Alu1.
===Electrophoresis===
* "SYBR". Small vial. Stain. Supplier: bugs
* SafeWhite nucleic acid stain. NBS Biologicals, cat NBS-SW1
* DNA Marker-D (100 bp-2000 bp). This is the "ladder" for electrophoresis comparisons. Supplied by NBS Biologicals, nbsbio.co.uk, cat: GM335
*1KB DNA ladder. Current ladder is aliquoted with loading dye from a 50 ug sample of concentration 0.2 ug / ul. [http://www.taq-dna.com/rich_files/attachments/DNA_Ladders_DNA_Weight_markers_DNA_leiter/1000_bp_1kb_DNA_Ladder.pdf key]. £36 + £15 shipping for 50ug (120 uses) from [http://www.nbsbio.co.uk/search.asp?s=ladder&Submit.x=0&Submit.y=0 nbsbio]
*Ethidium bromide stock solution - 2 ul ethidium bromide solution to 40 ml water.
*Powdered agarose (£46.80 + £15 shipping for 100g. Enough for 200 gels) from [http://www.nbsbio.co.uk/product.asp?pID=12205&cID=316 nbsbio].
*[http://www.nbsbio.co.uk/product.asp?pID=6241&cID=75 Safewhite 1ml]
*[http://www.nbsbio.co.uk/product.asp?pID=10571&cID=33 DNA Ladder]
*10x TBE - £33.60 for enough powder for 4L (enough for 267 50ml 1% TBE gels + 100ml 1% TBE buffer) from [http://www.nbsbio.co.uk/product.asp?pID=12335&cID=321 nbsbio]
*[http://wiki.london.hackspace.org.uk/view/Project:Biohacking/Stains List of possible DNA stains]


[[Category:Biohacking]]
[[Category:Biohacking]]
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