...concentration of puromycin should be from 1-10 μg/mL in 1 μg/mL increments. d. Label plates from 1-10 and add...of Contents A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium A.2 Map of pLKO.1 A.3 Related plasmids...Order oligos compatible with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing... References H.1 Published articles H.2 Web resources I. Appendix I.1 Sequence of pLKO.1 TRC-Cloning Vector...information Back to Top A. pLKO.1-TRC Cloning Vector A.1 The RNAi Consortium The pLKO.1 cloning vector is the backbone...Appendix I.1. Sequence of pLKO.1 TRC-Cloning Vector Click here to see the sequence of pLKO.1 TRC-cloning...the puromycin resistance marker encoded in pLKO.1 allows for convenient stable selection. Figure 1 : Map...

...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...you can transform 1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...Vector by Gel Purification Run your digest DNA on an agarose gel and conduct a gel purification to isolate...isolate the DNA. When running a gel for purification purposes it is important to have nice crisp bands and ...and grow overnight cultures for DNA purification. After purifying the DNA , conduct a diagnostic restriction...TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Experimental Procedure Run PCR and Ppurify the PCR Product Run PCR to amplify your insert ...

...you can transform 1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...vector by gel purification Run your digested DNA on an agarose gel and conduct a gel purification to isolate...isolate the DNA. When running a gel for purification purposes it is important to have nice crisp bands and...and grow overnight cultures for DNA purification. After purifying the DNA , conduct a diagnostic restriction...digest is a commonly used lab technique. For the purposes of this tutorial we will discuss how to move a...plasmids. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...