In-lab procedure You must set up your reactions according to your specific recipe. Don’t forget to digest the positive control (DS1-P, DS2-P for example) PCR reactions. Example enzymatic digest set up for two DS markers: DS1 SNP Marker Tube label 1 DS1-P 2 DS1-A 3 DS1-B 4 DS1-C 5 DS1-D 6 DS1-E DS2 SNP Marker Tube label 7 DS2-P 8 DS2-A 9 DS2-B 10 DS2-C 11 DS2-D 12 DS2-E Note: you do not need to digest the no-template negative control reactions. If you have already run the negative control samples on the gel described in lab 2.5, and confirmed an absence of product, those control samples are no longer needed. Alternatively, you can run the undigested negative control samples in the genotyping gel described later. Incubate your digestion reactions for 2 hours at the appropriate temperature. See the NEB catalog or website for information on your enzyme Lab Question 2: We are setting up these reactions for a complete digestion (no cleavable product remains after our incubation). Why is this important for our experiment? How would it hinder our analysis if only 50% of cleavable products were digested? During the digestion incubation, set up your gel apparatus and pour your genotyping gel as described in the next section. Genotyping electrophoresis gel and analysis of dCAPS markers In order to distinguish between the cut and uncut products, which are only different by ~20bp, we will need to run a high percentage (2.5%) agarose gel. Below are instructions for making 100ml of agarose gel in TBE buffer. Scale up or down according to the volume of your gel trays and the number of gels you will need.
In-lab procedure You must set up your reactions according to your specific recipe. Don’t forget to digest the positive control (DS1-P, DS2-P for example) PCR reactions. Example enzymatic digest set up for two DS markers: DS1 SNP Marker Tube label 1 DS1-P 2 DS1-A 3 DS1-B 4 DS1-C 5 DS1-D 6 DS1-E DS2 SNP Marker Tube label 7 DS2-P 8 DS2-A 9 DS2-B 10 DS2-C 11 DS2-D 12 DS2-E Note: you do not need to digest the no-template negative control reactions. If you have already run the negative control samples on the gel described in lab 2.5, and confirmed an absence of product, those control samples are no longer needed. Alternatively, you can run the undigested negative control samples in the genotyping gel described later. Incubate your digestion reactions for 2 hours at the appropriate temperature. See the NEB catalog or website for information on your enzyme Lab Question 2: We are setting up these reactions for a complete digestion (no cleavable product remains after our incubation). Why is this important for our experiment? How would it hinder our analysis if only 50% of cleavable products were digested? During the digestion incubation, set up your gel apparatus and pour your genotyping gel as described in the next section. Genotyping electrophoresis gel and analysis of dCAPS markers In order to distinguish between the cut and uncut products, which are only different by ~20bp, we will need to run a high percentage (2.5%) agarose gel. Below are instructions for making 100ml of agarose gel in TBE buffer. Scale up or down according to the volume of your gel trays and the number of gels you will need.
Hultman%20and%20Mellgren%202014%20Fetching%20SNPs%20Student%20Manual.docx