RT-PCR-DictyBase.docVIP

Dictyostelium RT-PCR for Knockout Screening and Expression Analysis The following protocol can be used to screen potential Dictyostelium KO mutants to confirm gene ablation, or for developmental expression studies. In mutant screening, we advise dilution plating of potential knockout mutants onto bacterial plates to ensure single Dictyostelium clones are used to test for loss of expression. 1. RNA isolation Below is a short description of rapid RNA isolation. Alternatively you can use commercially available kits. SDS Lysis buffer: 50 mM Tris Cl pH 7.5 0.1 M NaCl 10 mM EDTA 1% SDS harvest 2 x 107 cells wash with 1 ml KK2 the pellet can be stored at -80 °C (max amount of samples processed at once is 4 or 5 to reduce the time samples are stored in lysis buffer) re-suspend pellet in 500 μl lysis buffer by pipetting up and down add 500 μl phenol Incubate on a rotary shaker for 20 min, RT to ensure constant mixing centrifuge 5 min 13,000 rpm transfer the upper phase in a new tube and add 200 μl chloroform centrifuge 1 min 13,000 rpm transfer the upper phase in a new tube and add 1 ml 100% ethanol incubate 20 min at -20 °C centrifuge 5 min 13,000 rpm remove supernatant completely add (without further drying) 50 μl fresh Millipore water Measure the RNA content in a spectrophotometer (use a 1:50 dilution) Check RNA quality with a GTC gel (below) 2. GTC gel It is necessary to check for RNA degradation by visualising total RNA on a gel. To do this, prepare a standard agarose gel containing 5 mM Guanidine thiocyanate (GTC), as described. Two strong bands with minimal ‘smearing’ indicates intact RNA. Ethidium bromide is present in the loading solution, so do not add it to the gel. dissolve agarose in 1x TBE as for a DNA agarose gel prepared fresh 5 mM guanidine thiocyanate in 1 x TBE when the agarose is below 60 °C add 1 ml of the GTC solution (for 200ml gel) run gel as you would a DNA gel 3. Preparation of samples take 1 μl of your RNA sample and add 3 v