11 November 2013 2 683 Report

I am new to this and need to order for my experiment work. Please elaborate from the basics

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What are the steps for cloning a gene fragment in the pGEX-4T1 vector?

My DNA insert size is 746bp. I have designed the primers with the EcoR1 and Sal1 enzyme sites at 5'.

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How can one design the Cas9 gRNA for carrying out CRISPR/Cas9 mouse genome editing?

I just narrowed it down to the specific exon of gene of my interest but I am not sure how to make multiple gRNAs for the same gene target? What points should be kept in mind while designing the gRNAs?

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How to amplify a gene of 6.9 kb to clone in pDONR221 using Qiagen kit for reaction mix?

I used initial denaturation at 95 for 10 min followed by 94 for 45 sec. Temperature gradient for annealing temp from 62 to 73 at 1 min. Extension time : 68 for 6 min and further 68 for 10 min....

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