1,114
edits
Changes
Anorexia anx/anx mutant mice (view source)
Revision as of 10:58, 24 October 2024
, 10:58, 24 October 2024added some details
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===Gel===
−1% agarose in TAE (1 g agarose in 100 ml TAE, microwave 2 min at 50 power; add 10 ul of SYBR safe 10000x)
−load 10 ul of 50bp ladder, 10-20 ul of pre-digest and digest
* 2024-1-17 Tyro3 primers ordered from FSU BIO Core
* 2024-1-17 Tyro3 primers ordered from FSU BIO Core
−* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product-Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product-Information 50bp ladder]
+* 2024-1-18 [https://www.neb.com/en-us/products/r0126-nlaiv#Product-Information NlaIV] ordered from NEBiolabs, along with [https://www.neb.com/en-us/products/n0556-quickload-purple-50-bp-dna-ladder#Product-Information 50bp ladder] and purple dye.
* 2024-6-6 [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega GoTaq colorless master mix] ordered from Fisher (suspect green master mix interferes with digest)
* 2024-6-6 [https://www.promega.com/products/pcr/taq-polymerase/gotaq-master-mixes/?catNum=M7132 Promega GoTaq colorless master mix] ordered from Fisher (suspect green master mix interferes with digest)
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[https://omegabiotek.com/product-category/genomic-dna/cultured-cells-and-tissue/ E.Z.N.A. Tissue DNA Kit] yields 200 ul in elution buffer.
[https://omegabiotek.com/product-category/genomic-dna/cultured-cells-and-tissue/ E.Z.N.A. Tissue DNA Kit] yields 200 ul in elution buffer.
+
+Measure DNA quantity/quality with Nanodrop.
===PCR===
===PCR===
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PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).
PCR: 95C for 1min, then 30 cycles of: 95C for 1min, 72C for 2min. (note annealing and extension steps combined into 2 min at 72C).
+
+===Gel===
+
+2% agarose in TAE (2 g agarose in 100 ml TAE, microwave 2 min at 50% power; add 10 ul of SYBR safe 10000x)
+
+load 10 ul of 50bp ladder; 10 ul of PCR product + 2 ul purple loading dye
+
+run at 90V for 30 min
===PCR clean-up===
===PCR clean-up===
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===Digestion===
===Digestion===
−