Lab book of the gilestro lab
Protocols for molecular lab
Reagents needed and providers: Reagents Tris Hcl pH 7.5 VWR A4263.0500 £21.90 500ml PEG 8000 Sigma P1458-25ML £15.63 25ml MgCl2 Thermofisher AM9530G £29.70 100ml DTT VWR A3668.0050 £30.60 50ml NAD NEB B9007S £16.80 0.2ml dNTP Thermofisher 15363397 £49.50 1ml Phusion NEB M0530S £56.00 100U Taq Ligase NEB M0208S £49.60 2000U T5 exonuclease NEB M0363S £41.60 … Continue reading Gibson Assembly – DIY recipe
Instructions according to Stockinger lab manual: http://www.oardc.ohio-state.edu/stockingerlab/Protocols/GlycerolStocks.pdf Make a glycerol stock of plasmid pick a single colony from the transformation plate and grow an o/n culture in appropriate medium, e.g. LB amp put 0.5 ml of 80 % sterile glycerol in sterile screw cap microcentrifuge tube add 0.5 ml of o/n culture vortex freeze @ … Continue reading [Glycerol stocks] plasmids
Immunolabelling of larval and adult brains Dissect brains in PBS Fix with 4% formaldehyde in PBS containing 0.5 EGTA, 5mM MgCl2 (see below) @RT for 25 min. Wash 3 x 5 min. (approx.) in PBST (0.3% Triton-X100 in PBS). BREAK Can store in 4% PBT @RT or 4 DEG. 4) Preincubate in 90% PBST / … Continue reading [Protocol] Immunostaining protocol from Tony (Southall)
Some helpful websites and tools for some in silico research on fly genes FlyBase, http://flybase.org/ – Ties to together resources about the individual genes, including GBrowse gene locus viewer, predicted function according to InterPro and UniProt, lists of associated papers, etc. FlyAtlas 2, http://flyatlas.gla.ac.uk/flyatlas/index.html – Gene expression profile, based on in situ hybridization (reliability?) Virtual Fly … Continue reading Fly Resources
Gel Preparation: Below shows the volume of agarose solution required to make the desired agarose gel for each unit tray size. 1. For a standard 0.7% agarose gel, add 0.7 grammes of agarose to 100 ml of 1x TAE or TBE solution. The same 1 x solution should be used in the tank buffer solution. … Continue reading Gel volume/tank @ Cleaver Scientific for reference
Tested protocols – Phenol-Chloroform extraction from F Schweisguth @ http://francois.schweisguth.free.fr/protocols/Extraction_Drosophila_total_RNA.pdf – Chloroform extraction from FlyChip @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf – P Emery. RNA From Drosophila Heads, From: Methods in Molecular Biology, vol. 362: Circadian Rhythms: Methods and Protocols. E. Rosato [ed] © Humana Press Inc., Totowa, NJ Settled for the Emery Protocol for simplicity, yield and success. … Continue reading Trizol RNA Extraction Protocol: Emery Protocol w/ DNase I treatment
Protocol “Extraction of Drosophila total RNA” @ http://francois.schweisguth.free.fr/protocols/Extraction_Drosophila_total_RNA.pdf (extraction based on combination of Trizol and Phenol-Chloroform, spinning steps @ 4 DEG) Samples app. 12 male fly bodies and app. 30 heads, dissection on CO2 pad @ RT. Process Samples frozen @ – 80 DEG in PBS before extraction. Protocol as instructed, but used centrifuge at … Continue reading Phenol-Chloroform RNA extraction, Schweisguth Protocol
Protocol “Small scale extraction of total RNA from Drosophila melanogaster” @ http://www.flychip.org.uk/protocols/gene_expression/small_extraction.pdf (extraction based on combination of Trizol and Chloroform) Samples app. 30 male fly bodies. Process After incubation @ -20 DEG (step 12), sample was split in two. Sample 1 was temporarily stored @ -2 DEG in 500 ul ethanol, while sample 2 was … Continue reading Trizol RNA extraction of bodies, Flychip Protocol
Some general stuff For a reminder on how to calculate the volumes you need for the product you want to make, see, e.g., here: http://abacus.bates.edu/~ganderso/biology/resources/dilutions.html. If your buffer is based on powdered ingredients, add the powder first and add water up to your desired volume. Put your buffer in glass bottles to autoclave later (there … Continue reading Buffer recipes
From Georg Dietzl in Barry Dickson’s Lab, IMP Vienna 12/2002 SQUISHING BUFFER (SB) 10 mM Tris-HCl pH8 1 mM EDTA 25 mM NaCl 200 g/ml Proteinase K Procedure 200 g/ml Proteinase K are added to the squishing buffer (SB) freshly from 20X stock every day. Place 1-2 (I always take 2) flies in a 0.5 … Continue reading Single Fly Genomic DNA Prep
The lab uses a shared server for saving data. Data on the server are safe and go regular backup. You should map the server drive as network drive on your computer. When prompted, you should enter the login credentials for the network hard drive, which are: username: student password: ic_student Here is how to map … Continue reading Mapping Lab Server
The gel machine has now access to the college network and you can easily access the pictures you take in the lab from your own computer. Here is how to do it: If not logged in yet, login with local user “geldoc” ( password “edas%290” ). If you don’t have one yet, create a subfolder … Continue reading How to use the GELDOC computer in the molecular lab
https://boglab.plp.iastate.edu/protocols Includes: 1) Soft copy of the Golden Gate TALEN assembly protocol. http://www.addgene.org/static/cms/files/GoldenGateTALAssembly2011.pdf 2) an online tool with useful information about the plasmid sequences used in the Golden Gate TALEN kit and generation of sequences based on the RVD input. http://baolab.bme.gatech.edu/Research/BioinformaticTools/assembleTALSequences.html 3) an excel sheet that generates the reagents needed after you enter your RVDs. … Continue reading Useful tools for TALEN assembly
PBS 10X 40 g NaCl 1 g KCl 13.4 g Na2HPO4-7H2O 1.2 g H2O Adjust the pH at 7.4 with HCl. Complete to 500 mL with distilled water. Lysis Buffer 580 µL Nonidet P40 0.58 g CHAPS 0.150 g HEPES 0.508 g NaCl 9.93 g Saccharose 0.022 g Na2EDTA Adjust the pH to 8.0. Complete … Continue reading Western Blot Recipes
Make the RNA/primer/dNTP mix by combining the following components in a sterile RNase-free microfuge tube: Total RNA 1–10 μl (1 ng–1 μg) Primer dT23VN 2 μl dNTP Mix 4 μl nuclease-free H20 variable Total Volume 16 μl Heat for 5 minutes at 70°C. Spin briefly and promptly chill on ice. Add the following components to … Continue reading mRNA RT with ProtoScript® M-MuLV Taq RT-PCR Kit (NEB)
10X TAE Electrophoresis 48.4 g of Tris base [tris(hydroxymethyl)aminomethane] 11.4 mL of glacial acetic acid (17.4 M) 3.7 g of EDTA, disodium salt deionized water Dissolve the Tris, glacial acetic acid and EDTA in 800 ml of deionized water. Dilute the buffer to 1 L. You do not need to sterilize the solution. Store the … Continue reading Common Buffers – TAE
The following is a common method for the preparation of 1 liter of LB [source]: Measure out the following: 10 g tryptone 5 g yeast extract 10 g NaCl Suspend the solids in ~800 ml of distilled or deionized water. Add further distilled or deionized water, in a measuring cylinder to ensure accuracy, to make a … Continue reading Bacterial Growth solutions
NEB Recipe: 1X Gel Loading Dye, Blue (6X): 2.5 % Ficoll 400 11 mM EDTA 3.3 mM Tris-HCl 0.017 % SDS 0.015 % Bromophenol Blue pH 8.0 @ 25°C Simpler alternative Makes 100 ml. Store at room temperature (indefinitely). 0.25 g bromophenol blue (m.w. 669.96) 0.25 g xylene cyanol (m.w. 538.60) 50.00 g sucrose (m.w. … Continue reading DNA Loading Buffer
For reference.
When submitting sequences to MWG, follow directions as in the attached PDF document (here) Briefly, for plasmidic DNA from minipreps: Make sure your mini is of good quality, that is obtained with a Qiagen kit and of at least 200ng/ul (standard yield for kit preparation) Use 1.5ml eppendorf tubes with the proper sticky label on … Continue reading Sequencing in Tube format
Procedure Shake the culture tube to resuspend the cells. Label 2mL tubes and pour about 2ml of the cell suspension into each tube. Close the caps and place the tubes in a centrifuge (remember to balance the centrifuge by putting the tubes opposite one another) and spin at 11.000 rpm for 2 minutes. Withdraw and … Continue reading Plasmid miniprep
Batch makes about 40 plates. Making the LB Agar Add 250 mL of dH2O to a graduated cylinder. Weigh out 20g of premix LB Agar powder (VWR DF0445-17) or: 5.0 g tryptone 2.5 g yeast extract 5.0 g NaCl 7.5 g agar Mix powder well to bring into solution Add dH2O to total volume of … Continue reading LB Agar Plates
Topo TA cloning is done following loose suggestions of the manufacturer. We currently adopt the kit K204040 from Invitrogen, which is not based on topoisomerase ligation but regular T4 ligation. Comes with “One Shot® aTOP10 Chemically Competent E. coli”. Briefly. Quantify PCR product. The kit suggests to use 10ng of DNA. We usually skip this … Continue reading TOPO TA Cloning
Solutions: Solution A: [Tris HCI 0.1 M (pH 9.0), EDTA 0.1 M, SDS 1%] (keep at RT) Phenol-Cloroform: [If done fresh: mix Phenol and Cloroform 1:1, shake, spin for 10 min at 4.000 rpm] (keep at 4°C) KAc 8 M (keep at RT) Isopropanol (keep at -20°C) EtOH 70% (keep at -20°C) TE (keep at 4°C) … Continue reading Good quality genomic DNA extraction
Kit is NEB E5000-S ($95) Protocol as indicated by NEB. Use attached XLS file for help in creating the mix
Current Kit is N3232S [ 200μl, 200 gel lanes (500 μg/ml) ] To prepare a working dilution, dilute 30 μl in 120 μl TE1X, add 25 μl of Loading Buffer 6X. Use 6 μl of working dilution per well