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====Debugging plan (re-organized)==== *Re-planning from the previous debugging plan ([[Hosuk:LabNotes/2013-4-1|04/01]]) =====mRNA population check after fixing===== *mRNA were observed from the previous result([[Hosuk:LabNotes/2013-3-17|03/17]]), but just do again with the new samples fixed at 05/01 *Detail procedure *#Fix cells and permeabilize using 70% ethanol as before *#Mix 5 ul fluorescent primer (Cy3-polydT(50 mer)) in 200 ul 2xSSC/10% dextran sulfate/10% formamide *#Add to cells and incubate at 37C for 1 hour *#Wash using 2xSSC/10% formamide for 15 min each twice *#Aspirate and add 2x SSC and then image =====Reverse Transcript check===== *Test if RT process works well *Prepare two samples of fixed cell, and make one dish as a control without RTase *Detail procedure **Add 0.1N HCl for 10 min. **Wash with PBS twice **Prepare the reverse transcription mixture on ice (important). {| {{table}} | Reagent || | Volume |- |DEPC-H2O (Ambion) || |159uL |- |10x MMLV reaction buffer || |20uL |- |25 mM dNTP (Enzymatics) || |2uL |- |4 mM aminoallyl dUTP (Anaspec) || |2uL |- |100 uM /5Phos/TCTCGGGAACGCTGAAGANNNNNN || |5uL |- |RNase Inhibitor (Enzymatics) || |2uL |- |MMuLV reverse transcriptase (Enzymatics) || |10uL |} *#Add the enzyme cocktail to the fixed cells. ('''S2: +RTase, S3: -RTase''') *#Incubate at RT for 10 min, then transfer to 37Β°C for overnight. *#Aspirate. *#Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT. *#Wash with PBS once *#Block with 1M Tris pH 8.0 for 30 min. *#Wash with PBS once *#Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C. *#Rinse the entire well with ddH2O twice to remove traces of phosphate. *#Prepare 100uM of Cy3 labeled probe (Cy3-TCTTCAGCGTTCCCTAGA) *#2uL of 100uM probes in 200uL 2x SSC *#Pre-heated probe solution between 60C and 80C for 5min *#Add to cells at room temp, and let it cool down over 5min *#Wash with 2x SSC (2mL, but 200uL work too) twice and image! =====CircLigase II reaction on the dish incubating in the oven===== *CircLigase II reaction in a tube was confirmed ([[Hosuk:LabNotes/2013-4-22|04/22]]) *Test if CircLigase II reaction works well on the dish, and using 60C oven *Also, in Jay's protocol, minetal oil covering is used for preventing evaporation *Thus I need to compare two same CircLigase II mix on the glass bottom MatTek dish, but one dish is on the wet towel, another one is covered by mineral oil. *And then run a gel with two product by extract volume *Detail procedure **Prepare 50uL CircLigase II reaction mix **[[File:CircLigaseII-50uL.jpg|350px]] **Add 50uL CircLigase II mix in new, blank MatTek dish without any cells **One dish on wet towel, another one covered by mineral oil **Run 2hr. @ 60C in oven =====Rolony test with using pre-circulated DNA===== *This test was done at [[Hosuk:LabNotes/2013-4-24|04/24]], but try one more time. *Since I've suspected that the binding between BS(PEG)9 and aminoallyl dUTP doesn't work well, so the rolonies could be washed out while washing step between after RCA and before adding BS(PEG)9, *3 dishes will be prepared to check this. *Detail procedure **Prepare pre-circularized DNA using 20 uL reaction in the tube ***Two identical 20uL reaction ***12pmole of custom ssDNA(50bp) in each tube **Add pre-circularized DNA in 3 dishes (S4, S5, S6) ***S4 : 3uL of Linear ssDNA + 47uL H2O (6 pmole of DNA) ***S5 : 10uL of pre-circularized DNA + 40uL H2O (6 pmole of DNA) ***S6 : 10uL of pre-circularized DNA + 40uL H2O (6 pmole of DNA) **Run RCA **After RCA ***S4 : Aspirate, then wash with PBS once, then add BS(PEG)9 ***S5 : Aspirate, then add BS(PEG)9 ***S6 : Aspirate, then wash with PBS once, then add BS(PEG)9 **block with 1M Tris pH 8.0 for 30min. **Aspirate, wash with PBS once **Prepare 100uM of adapter sequence Cy5-TCTCGGGAACGCTGAAGA ***2uL of 100uM probes in 200uL 2x SSC ***Pre-heated probe solution between 60C and 80C for 5min ***Add to cells at RT, and let it cool down over 5min ***Wash with 2x SSC (2mL, but 200uL work too) twice, and image ====Result - mRNA check==== *A lot of mRNA were observed {| {{table}} | Bright field, 20x || | Cy3, 20x || | Merged || |- |([[Media:PolyA-FISH_fig04_20xobj_pos02_BF.tif|Original]]) || |([[Media:PolyA-FISH_fig03_20xobj_pos02_Cy3.tif|Original]]) || | |- |[[File:PolyA-FISH_fig04_20xobj_pos02_BF.jpg|300px]] || |[[File:PolyA-FISH_fig03_20xobj_pos02_Cy3.jpg|300px]] || |[[File:Composite_fig03-04_2013-05-014.jpg|300px]] |}
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