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# Tail digest DNA
*[[PCR Amplification of DNA]]
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#Resuspend DNA in 6 mL of distilled water. Add 10g of CsCl and dissolve.
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# Select '''Enter Individual DNA Sequencing Requests'''
* 1 uL of template (unless the plasmid DNA concentration is >500 ng/uL in which case dilute it to 50-300 ng/uL
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This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been comp
...sion or use the shaker. '''NEVER''' vortex samples, as it will destroy the DNA.
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[[Category:DNA]]
===Yeast DNA Prep Buffer===
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[[Category:DNA]]
...e Yeast. See [[Rapid Isolation of Genomic DNA from Yeast]]. Use 1µl of the DNA in your PCR reaction.
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===DNA Extraction===
** 1 uL of the Extracted DNA from the previous step.
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*Calculate DNA to transfect. Start with 4 ug per 6 well
*Lipofectamine(in uL) = DNA(in ug) * 2.5
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*Calculate DNA to transfect
*Lipofectamine(in uL) = DNA(in ug) * 2.5
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* DNA - typically transfect 50-1000 ng DNA per well
##Per ug of DNA need 3 uL Fugene.
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* D-Loop Primers (for determining the number of mitochondrial DNA copies)
* DNA Primers (for determining the number of nuclear DNA copies). Can use any genotyping primers with an amplicon <200bp. We use t
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# Tail digest DNA
*[[PCR Amplification of DNA]]
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773 bytes (111 words) - 17:00, 8 June 2020
===DNA Extraction===
** 1 uL of the Extracted DNA from the previous step.
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*OptiMEM(in uL, equal for DNA and Lipofectamine) = Lipofectamine(in uL) * 50
##Combine the two volumes of OptiMEM/DNA and OptiMEM/Lipofectamine (should be 200uL/well).
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# Select '''Enter Individual DNA Sequencing Requests'''
* 1 uL of template (unless the plasmid DNA concentration is >500 ng/uL in which case dilute it to 50-300 ng/uL
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*DNA of interest (1 uL for plasmid, 5 uL for cloning/mutagenesis)
#Add DNA to cells and mix by tapping
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[[Category:DNA]]
...e Yeast. See [[Rapid Isolation of Genomic DNA from Yeast]]. Use 1µl of the DNA in your PCR reaction.
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[[ Category:DNA]]
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* Prepare DNA in a sterile 1.5 mL tube or tubes as needed. Use 100 uL per well.
* Add 3 uL PEI per ug of DNA in OptiMEM. The total volume should be 100 uL per well. if transfecting m
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...our region of interest spans several introns, you need to copy the genomic DNA not the mRNA sequence. In other words, if you can design your CRISPR pairs
* Based on where you want to cut the DNA select the one or two guide DNA sequences. Name those sequences based on the nucleotide it cuts after (see
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*GST-HA-S6K1 plasmid. Prepare by [[Cesium Chloride Preparation of DNA]]. Need 500 ug per 10 plates of cells
#Combine 500 ug DNA with 10 mL OptiMEM, and separately 700 uL Lipofectamine 2000 with 10 mL Opt
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1 KB (185 words) - 19:59, 20 September 2012
This protocol is for extracting DNA from TRIZOL preparations after the RNA-containing upper phase has been comp
...sion or use the shaker. '''NEVER''' vortex samples, as it will destroy the DNA.
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3 KB (417 words) - 17:42, 25 September 2015
[[ Category:DNA]]
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[[ Category:DNA]]
...e targeted allele only. For Southern blotting, SalI/EcoRI-digested genomic DNA was probed with a 0.4-kb fragment immediately upstream of the 5′ arm (Suppl
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*Sample Genomic DNA (extracted as described in [[Blood DNA Extraction]])
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* Prepare DNA according to this table in a sterile 2 mL tube:
* For this amount of DNA (28 ug) try using 3:1 PEI:DNA, so add 84 uL PEI into 1 mL OptiMEM.
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[[ Category:DNA]]
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