AfaI (RsaI) restriction enzyme

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The other three libraries were each made from DNA digested with three different restriction enzymes; AluI (Fermentas), HaeIII (TaKaRa Bio Inc.) and AfaI (TaKaRa Bio Inc.).	Each digested DNA was purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA) and ligated with double stranded DNA linkers (linker 1: 50 -GTTTAGCCTTGTAGCAGAAGC-30 and linker 2: 50 -pGCTTCTGCTACAAGGCTAAACAAAA-30 ).

Protocol tips
The other three libraries were each made from DNA digested with three different restriction enzymes; AluI (Fermentas), HaeIII (TaKaRa Bio Inc.) and AfaI (TaKaRa Bio Inc.).
Downstream tips
Each digested DNA was purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA) and ligated with double stranded DNA linkers (linker 1: 50 -GTTTAGCCTTGTAGCAGAAGC-30 and linker 2: 50 -pGCTTCTGCTACAAGGCTAAACAAAA-30 ).

Publication protocol

An enriched genomic DNA library was constructed from Huay Bong 60 as described by Nunome et al. (2006) with some modifications. Leaf tissue was collected for DNA extraction using DNeasy Plant Mini Kit (QIAGEN, Hilden Germany). Four libraries were constructed. For the first library, 20 lg of genomic DNA was digested with two different restriction enzymes; AluI (Fermentas, Hanover, MD, USA), HaeIII (TaKaRa Bio Inc., Ohtsu-shi, Japan). The other three libraries were each made from DNA digested with three different restriction enzymes; AluI (Fermentas), HaeIII (TaKaRa Bio Inc.) and AfaI (TaKaRa Bio Inc.). Each digested DNA was purified using Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA) and ligated with double stranded DNA linkers (linker 1: 50 -GTTTAGCCTTGTAGCAGAAGC-30 and linker 2: 50 -pGCTTCTGCTACAAGGCTAAACAAAA-30 ). Ligation reactions were performed in 60 ll reactions containing 10 lM DNA linkers, 19 rapid ligation buffer, 24 U of T4 DNA ligase (Promega), 20 U XmnI (Promega), 10 U of each restriction enzyme in individual reaction: AluI, HaeIII and AfaI. Ligation reactions were accomplished by 25 cycles of 30 min at 16C, and 10 min at 37C, and incubated at 16C overnight.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Restriction Enzymes RsaI / AfaI using AfaI (RsaI) restriction enzyme from Takara Bio Inc.

Manufacturer protocol

Download the product protocol from Takara Bio Inc for AfaI (RsaI) restriction enzyme below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Restriction Enzymes RsaI / AfaI using AfaI (RsaI) restriction enzyme from Takara Bio Inc. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.