DNA Topoisomerases by Marc Drolet

Author:Marc Drolet
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

2.Use a standard procedure to amplify by PCR a spacer DNA region to position the ssDNA bulge at the desired distance from the Dig handle. The spacer would have ApaI and SacI restriction sites on either end. After digestion with only SacI, purify the spacer using a PCR purification kit. Typically, we start with 100 μg of spacer, since multiple purification steps cause significant loss of product.

3.Purchase oligonucleotides with at least 20 bp of overlapping complementary regions on either side of the desired bulge, which is located in the middle of one of the oligos. For a 27 nucleotide (nt) bulge we use the sequence 5′-AATCGGCATTGCGCAAACCAAGACAG-3′ and for a 12 nt bulge 5′-AACGTGCCAAGA-3′ [9]. The oligonucleotides should have overhanging sticky ends that allow for ligation to the digested spacer (SacI) and the long linearized DNA (XmaI) respectively. The SacI overhang for the annealed oligonucleotides needs to be 5′ phosphorylated; we purchase the oligonucleotides prephosphorylated. Anneal the oligonucleotides by heating in a heat block to 95 °C for 3 min and allow cooling to room temperature (RT) in the heat block in annealing buffer (see Note 4 ).

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