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<div dir="ltr">Hi, I have been having problems with my 5-FOA Selection. I am trying to knock out a section of a gene in S.cerevisiae through a pop-in/pop-out method. I initially inserted an Ura3 gene into the region I wanted to delete and made the construct (I) with the URA3 gene. This construct (I) was made on a background strain BY4741 clean del Ura3Knockout replaced with a Kan marker (on a BY4741 WT). I then used this strain to have my clean knockouts by transforming PCR product homologous to the flanking region of the Ura3 gene that was inserted through the FOA selection method. This way I hoped to achieve a clean Knockout- Construct(II). But unfortunately, I could not get the second stage of FOA selection to work. I tried changing the protocol conditions (LiAc transformations) several times i.e using DMSO to achieve increased transformation efficiency etc but all went in vain. I get few colonies everytime (about 30-100) but all of them seem to be some sort of point mutants to construct(I), also I get twice the number of colonies in my control (no insert DNA) -(where I shld not see any colonies) as to the transformation plate. I recently tried electroporation and eventually selecting them on a FOA plate but they dont seem to work either (a positive control does work on both methods), all I end up getting eventually were point mutants and colonies in my control. I have tested my construct (I) and also the FOA plates that I use (by streaking up the construct (I) and observing for growth for 4-6 days) and they all look perfectly fine. I use 1gram 5-FOA/1000ml media adding it after autoclaving the media and cooling to about 50C. I have also tried using higher % of 5-FOA. Can someone help me find out if I need to do any alteration in my protocol or is there any other efficient method to get my construct (II). The pop-out insert is about 80bp in size. What could possibly be wrong here? Is there any well tested method that one could suggest for such a selection? I will be grateful for any help on this. Thanking you Regards, - ash# -----> The transformation protocol I use normally 1) Grow cells to exponential phase 2) RSP in 450ul LiAc and store O/N at 4C 3) Make the following solutions 100ul Cells 10ul CT DNA 10ul DNA(transforming) 700ul LiAc/PEG 4) Incubate for 30min @30C 5) Incubate for 20min@20C 6) Centrifuge for 5 mins at 6K 7) Wash cells in water 8) RSP in 1ml YPD 9) Incubate for 6hrs @ 30C 10) Wash cells in water 11) RSP in 300ul water and spread in plates. Additionally I have tried: after step 3) Incubate at 30C for 60 minutes 4) add 40ul DMSO mix thoroughly 5) Heat shock at 42C for 15 mins 6) Microfuge for 10sec resuspend in 1ml 1XTE (twice). 7) plate on 5-FOA media In Electroporation I used the following protocols as suggested here <a href="http://iubio.bio.indiana.edu:7131/bionet/mm/yeast/1996-July/005543.html">http://iubio.bio.indiana.edu:7131/bionet/mm/yeast/1996-July/005543.html</a> 1°) from Abhijit Datta (Abhijit_Datta at <a href="http://macmailgw.dfci.harvard.edu">macmailgw.dfci.harvard.edu</a>) OK...for maximal efficiency use the published protocols... 1) Harvest exponentially growing cells from 50ml culture (approx. 1X10e6). Wash 3 times with distilled water (chilled). Wash 2X with 10% glycerol (chilled). Resuspend in 50-100ul vol. <div class="western" id="g8t952" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify"> </div> <div class="western" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify">2) Make clean DNA by precipitating and washing 3X. OR use Qiagen preps. </div> Dilute DNA 10-100 fold (compared to LiAC protocol usage) in 10% glycerol. Add 5ul max DNA vol to 40ul cells in a chilled 0.2mm disposable cuvette. Set controls at 1.5Volts pulse, 25 for capacitance and 200 for resistance. (the last 2 same for coli transform using same cuvettes). <div class="western" id="g8t961" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify"> 3) Wash out cuvette with 500ul cold 10% glycerol immediately and plate</div> <div class="western" id="g8t965" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify">Immediately </div> <div class="western" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify">and -- </div> <div class="western" style="MARGIN-BOTTOM: 0.14in; LINE-HEIGHT: 100%" align="justify">from Pierre Falson (falson at <a href="http://verlaine.saclay.cea.fr">verlaine.saclay.cea.fr</a>)</div> taking a 3 weeks-less-old colony, growth in 100 ml Yeast Extract 1%, bactopeptone 1% and glucose 1%. Go to 1.3-1.5OD600nm/ml (10E8cell/ml). Centrifuge in two falcon tubes two times at 5000 rpm and wash with 100 ml of sterile, cold water, centrifuge, wash with 50 ml water, centrifuge, suspend in 4 ml 1 M sorbitol (sterile, cold), centrifuge, suspend in 0.1 ml of 1M sorbitol (s, c). Let the yeasts at 4°C. they can be used immediately or one or two days after. yeasts are aliquoted in 40 µl fraction, mixed with 0.1µgDNA/0.1-5µl buffer (TE). the mix is let on ice for 4 min. the apparatus (biorad) is setted to 1.5kV, 25 µF (gene pulser) and 200 Ohms (pulse controller). pulse in a 2 mm chamber (time constant is 4.5 - 5 msec). add 1 ml of 1 M cold sterile sorbitol, pour on plate. Colonies will appear in 48-72 h. I guaranty this protocol! Pierre Falson -- Ahamarshan.JN Department of Biochemistry University of Oxford Oxford OX13QU Ph:07809705559 </div>

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