He Lab Protocols
Mouse genetics
Mouse Genotyping Quick DNA isolation for genotyping Clean DNA isolation with choloroform prep Toe clipping protocol, Ear punch protocol Kras LSLG12D p53 p53 CKO allele Eu-Myc miR-34a KO miR-34b/34c KO miR-449 KO miR-34b/34c fl allele miR-200c/141 KO; miR-200b/200a/429 KO; miR- 200c/141-LacZ fl allele miR-200c/141:LacZ FLP'd OUT mTmG LSL-Cas9 (From Jacks Lab) dCas9-KRAB Mouse models Eu-myc B-lymphoma model fetal liver collection adoptive transfer KrasG12D induced lung adenocarcinoma model determine Ade-Cre viral titer intranasal incubation of adeno/lenti virus Intratracheal injection of lenti/Adeno virus Lung sample preparation - Histology Prepare primary lung cancer cell lines Teratoma generation from ESCs or iPSCs Superovulation for embryo collection CRISPR-EZ [protocol] [video] B6 colony Histology / IF / In situ / staining analyses HE using paraffin section KOS machine processing Bone paraffin embedding IF using cryo section - [Jenn] [Yongjin] IF using paraffin section [Trilogy] [No Trilogy] IHC using paraffin section mRNA/ncRNA in situ using cryo section mRNA/ncRNA in situ using paraffin section miRNA in situ using cryo section miRNA in situ using paraffin section TRAP staining PAS staining Trichrome staining EM preparation Molecular Biology General intro on RNA, DNA and protein work RNA biology RNA prep (trizol) microRNA real-time PCR He Lab protocol - Paul Lin ABI protocol mRNA real time PCR Single embryo/cell real time PCR [ABI kit] [Smart- seq protocol] Luciferase assays (for miRNA target validation) small RNA sequencing RNA-seq (polyA enriched, strand specific) -Li Lab small RNA northern [Hannon Lab] sgRNA design /in vitro transcription / purification CRISPR-editing in 368 lung cancer cell lines mRNA Northern Hammerhead ribozyme 5'RACE 3' RACE [Andrew's protocol] [Clontech protocol] Protein analysis Chromatin Immunoprecipitation (ChIP)- Keji Zhao CHiP data analyses -Paul Western (with tips for miRNA target studies) Bradford analysis - Frank Qi Western protocol - Paul Lin How to run a best western _Paul Luciferase assay for promoter activity Luciferase assay for translation efficiency Southern [Hannon lab] OP Puro Cloning Golden Gate cloning Gibson assembly Mutagenesis protocol - Enbo Ma Pfu Turbo DNA analyses Bisulfate sequencing [EZ DNA methylation kit], [Jump Start Taq] |
Cell Biology
Primary cell culture / derivation MEF derivation Mouse MEF culture/splitting/freezing Mouse tail fibroblast derivation Mouse primary B-cell culture [Em Ho] [Schlissel Lab] Lung adenocarcinoma cell derivation Lymphoma/ Leukemea cell derivation, culture, passage and freezing (Eu-myc, B-ALL) 3D culture of epithelial cells (or tumor cells) Matrigel testing Stem cell culture / derivation Mouse ESC derivation from blastocysts [Paul Lin Protocol] (recommended) [Sang Yong Kim protocol] [Manipulating mouse embryo protocol] [Earnest Arenas protocol] Mouse ESC/iPSC culture, passage, freezing Mouse TSC derivation from blastocysts Rossant's Lab three protocols [1] [2] [3] Mouse TSC culture, passage and freezing Mouse iPSC generation from MEF human iPSC generation from dermal fibroblasts MEF feeder preparation ESC transfection with RNA oligo ESC transfection with PiggyBac More reading on ESC transfection methods ESC transduction with lentivirus/retrovirus More reading on ESC lenti transduction Differentiation assays EB differentiation from mouse ESCs/iPSCs XEN cell differentiation from mouse ESCs/iPSCs EB differentiation from human ESCs/iPSCs Osteoclast cell differentiation Retroviral / lentiviral infection and transfection Pheonix cell culture/selection / splitting/freezing Pheonix cell selection Retrovirus production/infectionng Lentivirus production/infection IF / in situ / staining in cell culture mRNA/ncRNA in situ using cell culture miRNA in situ using cell culture IF in cell culture (ESCs) - Yongjin Choi PI / 7AAD staining BrdU staining Imaging experiment SIM staining STORM staining TEM and SEM sample preparation Reagents Home-made Competent cells Home-made Trizol [Mike Thompson] Home-made Taq [Mike Thompson] Taq testing protocol Paul Home-made LIF [Tijian Lab] Home-made Antifade Solution [Bin Xue] Recipes for home-made buffers Serum testing Matrigel testing Western ECL reagents [Mike Thompson] |