TIANamp Genomic DNA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

Plasmids construction
Genome DNA was isolated from human HEK293 cells. Fragment of 5′ upstream region of DYRK1A isoform 1 was amplified by PCR with primers (5′-CCGCTCGAGGATGATTGGGATGACATCATG-3′ and 5′-CCCAAGCTTCCAGCGGCAAAACTATAAC-3′) and cloned into pGL3-basic vector to make pDYluc-A construct. pDYluc-B construct was obtained from blunt end ligation of products of double digestion of pDYluc-A by EcoRI and XhoI. pDYluc-C and pDYluc-D were obtained by PCR using primers (5′-CCGCTCGAGTAGTTGATTTTGATTATTG-3′, and GLprimer2; 5′-CCGCTCGAGGAATGTTAGAAAATGAA-3′, and GLprimer2) with pDYluc-A as template. The restriction enzymes to generate pDYluc-C and pDYluc-D were XhoI and HindIII. Fragment amplified with primers (5′-CTAGCTAGCGAAACTGGCGAGGTGTAAGTAGCAT-3′, 5′-CCGCTCGAGTCATCCTGCTTAATAATCCTTTTCC-3′) were ligated with insert from pDYluc-A construct. The ligation was double digested by NheI and HindIII and then cloned into pGL-3 basic vector to generate pDYluc-long. Forward primers used for pDY-MRE and pDY-MREmut were 5′-GGGGTACCAATATATTTTATATATAGTTG-3′ and 5′-GGGGTACCAATATATTTCGGCCGGCAGTGATTTTGATTAT-3′ with KpnI as restriction site, respectively. They share the same reverse primer: 5′-CCGCTCGAGTCCAGCGGCAAAACTATAAC-3′ with XhoI as restriction site. They were both cloned into pGL3-basic as well. All the constructed plasmids were confirmed by DNA sequencing. Our deletions of plasmids were sequenced by forward primer RVP3 (5′-CTAGCAAAATAGGCTGTCCC-3′) supported by BioSune Biotechnology Inc. (Jinan, China).

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Manufacturer protocol

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