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Lab Notebook

May M T W T F S S 22 23 24 25 26 27 28 29 30 31 June M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 July M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August M T W T F S S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

May 22 Emily - Optimize the procedure to incorporate (yeast) KanMX, prefix, and suffix into a backbone containing yeast homologies. Add Mfel site and prefix to KanMX (PCR 1) via PCR. Gel verification (good results). PCR tubes were combined, purified and nanodrop for concentration. May 23 Emily - Adding a suffix, Mfel and product of PCR 1. Use the same annealing temperature, time, and PCR protocol since the fragment lengths are approximately the same. The PCR tubes were combined, purified, and the concentration was measured. The PCR product as well as the plasmid (IAde2 PSB1K3) were digested with MfeI-HF and PstI-HF at 37°C overnight. May 24 Emily - Add phosphatase to the IAde2 PSB1K3 digest to dephosphorylate the 5’ end and prevent recircularization. Incubate at 37°C for 30 minutes. Heat shock both digest solutions at 80°C for 20 minutes. Ligate IAde2 PSB1K3 and the KanMX insert. Incubate ligation mixture at room temperature for 2 hours, andthen heat shock at 65 degrees for 10 minutes. E.coli transformation. Victoria - Amplify yeGFP insert with primers pp22 and pp23, amplify yeGFP plasmids with a different set of primers. Gel verification (poor results). George - Plan steps for Gibson Assembly. May 25 Emily - Plates taken out. Good growth. May 27 Emily - Inoculate 12 (half) colonies as well as 2 negatives in 3mL of LB Kan at 37°C for 6 hours. Also inoculate 4 tubes of PSB1K3 IAde2 homo, IAde4 homo, IGal4 homo, and 3His3 homo in 3mL of LB Kan to replenish DNA stocks. Plate glycerol stocks of PSB1K3 IAde2 homo and KanMX::EXSP on LB Kan to create negative and positive controls for the colony PCR tomorrow. Miniprep the 12 inoculations. George - PCR from stock DNA solutions to create the desired fragments for Gibson Assembly. PCR: His3 pSB1K3 Backbone, Matts eGFP and pGal4 Backbone. Gel verification, poor results for pGal4 and good results for the other 2 PCR amplifications. May 28 Emily - The PSB1K3 IAde2 homo, IAde4 homo, IGal4 homo, and 3His3 homo inoculations grew, but it took a long time (it still looked clear after 6 hours yesterday). Miniprep and double check the contents of the stock with PCR. Gel verification (good results). Additionally, perform a colony PCR using the other half of the colonies from the plates, as well as the colonies from the Ade2 homo and Ade2::KanMX EXSP PSB1K3 plates that were made yesterday. Gel verification (null results). Gel digest and PCR products (usable results) and null results. George - Perform a yeast colony PCR today to try and isolate some fresh pGal4 using the IR163 strain. Redo of PCR amplifications. Gel verification (poor results). May 29 Emily - KanMX-MfeI-EXSP should be digested with MfeI and PstI, Ade2 PSB1K3 should be digested withEcoRI and PstI. However, it was digested with MfeI and EcoRI create the same sticky ends, but recognizedifferent cutsites, allowing the restriction sites to be eliminated. Digest Ade2 PSB1K3 with EcoRI-HF and PstI-HF. Incubate the Ade2 digest at 37°C for 2 hours. Add phosphatase to the Ade2 PSB1K3 digest to dephosphorylate the 5’ end and prevent recircularization. Incubate, Heat shock, Ligate the digests. E.Coli Transformation. Victoria - PCR all plasmids that contains yeGFP. His3::NatMX-yeGFP, Ade2::KanMX-yeGFP, Gal4::His3-yeGFP, Ade4::Ura3-yeGFP. Gel verification (usable results). Taylor - Digested Ade 2:: Kan MX-pSBIK3 , His 3::Nat MX-pSB1K3, Ade 4:: Ura 3-pSB1K3, Gal 4:: His 3-pSB1K3, and RFP-pSB1C3 with EcoRI and SpeI May 30 Emily - Decent growth on plate. Inoculation, 13 colonies, without colony PCR. Miniprep. Taylor - Added phosphatase to the digested Ade 2::Kan MX-pSBIK3 , His 3::Nat MX-pSB1K3, Ade 4:: Ura 3-pSB1K3, and Gal 4:: His 3-pSB1K3. Ligated Kan MX-pSBIK3 , His 3::Nat MX-pSB1K3, Ade 4:: Ura 3-pSB1K3, and Gal 4:: His 3-pSB1K3 with RFP-pSB1C3. May 31 Emily - Nanodrop the inoculations and digest 500ng of each tube as well as a positive with PmeI. Gel verification (usable results). Victoria - Digest of Gal4::His3-yeGFP, Ade4::Ura3-yeGFP, Ade2::KanMX-yeGFP, His3::NatMX-yeGFP withPmeI. Gel verification (poor results). Taylor - No visible growth on transformation plates. Gel digest to assure that all was properly digested (suspect that possibly the RFP 1C3 did not digest). June 01 Victoria - Digest of His3::NatMX-yeGFP, Gal4::His3-yeGFP, Ade2::KanMX-yeGFP, Ade4::Ura3-yeGFP with pmel. Gel verification (good results). June 03 Emily - Finding parts that can be improved from kit plates. Taylor - Miniprepped pSB1A3/RFP, His 3;;Nat, Gal 4;;His 3, and Ade 4;; Ura. Nanodrop the miniprep.Digested the minipreps. June 04 Emily - Transform the BioBrick from the 2017 kit plate and plate 100µL, 200µL, and a negative on LB CM. Victoria - Streaking Strains from BY4742 and W303a. Taylor - Added phosphatase to digests from Monday,June 3rd. Ligated all homologous platforms with RFP (including Ade 2;;Kan which was not miniprepped) (same ligation math as last ligation). June 03 rd. Ligated all homologous platforms withRFP (including Ade 2;;Kan which was not miniprepped) (same ligation math as last ligation). June 05 Emily - No growth. Streaked a glycerol stock of RFP PSB1C3 on a CM plate to incubate at 37°C overnight. Victoria - Prepared for sequencing of the plasmids His3::NatMX-yeGFP, Gal4::His3-yeGFP, Ade2::KanMX-yeGFP, Ade4::Ura3-yeGFP. Taylor - No growth on plate. Streaked LB Kan and LB Clm glycerol stocks. Gel digests (good results), confirmed biobricks. E.coli transformation. June 06 Taylor - Attempted to re-digest (heated up water bath to 42 degrees instead of using 42 degree incubator because it was found that this was too hot and potentially killing cells). Ligation. E.coli transformation. No growth. June 07 Victoria - Made plates. Inoculated 5 colonies of BY4742 strain. June 08 Victoria - Yeast transformation. Obtained data back from sequencing and none of them have yeGFP insert. June 09 Victoria - Yeast imprinting. Failed growth on plates. June 13 Emily - Start troubleshooting the transformation protocol. Try to digest Setti’s plasmid (Ade2::KanMX EXSP PSB1K3) and RFP PSB1A3 with SpeI and EcoRI-HF. Add phosphatase to the backbone (Ade2:KanMX). Heat shock. Ligate. Transform. June 14 Emily - Decent growth. Optimize the procedure to incorporate (yeast) KanMX, prefix, and suffix into a backbone containing yeast homologies. Add phosphatase to the IAde2 PSB1K3 digest with MunI and SpeI to dephosphorylate the 5’ end and prevent recircularization. Incubate. Heat shock digests. Ligate IAde2 PSB1K3 and the KanMX insert. Incubate ligation mixture at room temperature for an hour, and then heat shock at 65°C for 10 minutes. Victoria - Amplifying GFP seeding. Gel verification (one has worked). June 15 Taylor - Synthesized primers, prefix-pSB1K3-suffix and prefix-RFP-suffix. Transformed GFP open reading frame (for characterization). June 17 Emily - Transform the ligation mixture from Friday on LB kan. Streaked glycerol stock of any part in PSB1K3 on the new LB kan plates to ensure that the new tryptone. Research Biobrick to improve. Transforming the DNA from the 2019 kit plate 1, well 19E on LB CM. Victoria - Amplifying GFP seeding. Gel verification (poor results). Digest PCR GFP seeding with NotI. June 18 Emily - All plates grew. The glycerol stock test on the new LB kan plates grew, which means that the new tryptone is usable. The plate with the kit plate DNA had very good growth, whereas the plate for the standard assembly optimization protocol only had approximately 6 colonies on it. Victoria - Gel verification of GFP seeding digest (poor results). Taylor - Inoculated transformation from Friday. June 19 Emily - Inoculated 3 colonies with the kit plate DNA in 5mL LB CM, and 6 colonies from the MunI optimization test in 3mL LB Kan for 6 hours, and then miniprep. Victoria - PCR of GFP seeding with a gradient of lowest 60 to highest 70 Celsius. Gel verification(usable results). The best annealing temperature is 64 Celsius. Purification. PCR yeGFP insert amplification. Gel verification (good results). June 20 Emily - Digested 500ng from each Ade2::KanMX tube with PmeI and incubated at 37°C for 2 hours. Gelverification (poor results). Victoria - Purification and nanodrop. Digest Ade2:KanMX-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP, His3:NatMX-yeGFP for backbone with NotI. Digest the purified yeGFP. June 21 Emily - Gel the tubes again after the heat shock using 10uL of DNA in the small cassette (instead of the large cassette) (poor results). Digest using 1000ng of DNA via PmeI. Heat shock. Gel verification(poor results). Gel order: 10uL for all except 5uL undigested 2, no undigested 4, 5uL undigested positive. Gel verification (poor results) show that there is no incorporation of insert. Victoria - DNA ligation of the digests from yesterday. E.coli transformation. June 22 Victoria - Only Gal4:His3-yeGFP had a bit of colonies that can used to be verified with colony PCR. June 24 Emily - Miniprep Ade2 PSB1K3 and Ade2::KanMX PSB1K3 inoculations from Sunday. PCR prefix and suffix on RFP PSB1A3 using a gradient between 66°C and 70°C. Gel verification (poor results). Victoria - Colony PCR verification. Inoculate 5 tubes of 1 colony in each. June 25 Emily - Prepared primers for the amilCP improvement. Amplified URA3 to the GPD promoter (plus amilCP homo). Gel verification (good results). PCR purification and nanodrop. PCR the CYC1 terminator (plus amilCP homo) - URA3. Gel verification (good results). Victoria - Miniprep of Gal4:His3-yeGFP. Digest miniprep of Gal4:His3-yeGFP with NotI. Gel verification (poor results). Taylor - Prepped primers. Preformed 6 PCR amplifications. June 26 Emily - PCR purify and nanodrop the DNA. PCR GPD homo - amilCP CYC1 - terminator homo. Gel verification (good results). PCR purification and nanodrop. PCR Ura3 - GPD promoter - amilCP homo (longer) using yesterday’s PCR product. Gel verification (poor results).PCR amilCP homo (longer) - CYC1 terminator - URA3 UTR. Victoria - Redid a PCR since the one done did not show the yeGFP bands. It can be due to the fact that only 50ng of DNA was used rather than 100ng. The new PCR had 100ng of DNA.4 sets of Minipreps were done for the plasmids Ade2::Kan, Ade4::Ura3, His3::Nat and Gal4::His3 from glycerol stocks that were inoculated overnight. June 27 Emily - Gel verification (poor results). PCR GPD homo (longer) - amilCP - CYC1 terminator homo (longer). Gel verification (poor results). Redo PCR for URA3 - GPD promoter - amil CP homo (longer) using alarger temperature gradient. Gel verification (good results). PCR purification and nanodrop. Inoculate 2xYPD with adenine and yeast colony to prepare for yeast transformation. Victoria - PCR yeGFP seeding due to low concentrations. Gel verification (poor results). June 28 Emily - Yeast transformation on -Ura plates. Victoria - PCR yeGFP seeding due to low concentrations. Gel verification (poor results). New plan.Digest the 4 plasmids with EcoRI and SpeI. RFP inserts obtained from PSB1K3. Ligation of the insert andbackbone. E.coli transformation. Did George’s Transformation of 2 tubes: pHisPara2+T5 and pHisPara2+Taq(single plate for each + neg). Taylor - Preformed 4 PCR amplifications. July 02 Emily - Decent growth but the colonies aren’t blue. Check colonies via yeast PCR. Gel verification(usable results). Made a patch plate: take the other half of the colony and scrape it on a patch of a -Ura plate. Leave to incubate at 30°C. Resume optimizing the standard protocol, starting with inserting RFP into Ade2 PSB1K3 (and the other homologies). Incubate. Heat shock. Ligate homologies and RFP. Victoria - The colony of Ade2::Kan-RFP did not grow any colonies and the ones for George didn’t grow at all.Ligate RFP digest with Ade2:KanMX-EXSP pSB1K3, heat inactivated and transformed on Kan. Inoculate ½ red colony from all 4 plasmids in 5mL LB Kan for 6 hours and did a miniprep. Other ½ a colony PCR.Miniprep the 3 plasmids; Ade4::Ura3-RFP, His3::Nat-RFP, Gal4::His3-RFP. Taylor - Preformed PCR amplification. July 03 Emily - E.coli transformation. PCR colonies 4, 5, 6, 8, and 10 yesterday using the first primer (Ura3 forward) and second-last primer (CYC1 terminator reverse) since the last primer’s melting temperature is too different from the first primer. Gel verification (good results). Victoria - Colony PCR and inoculation of Ade2::Kan-RFP which is 2 tubes of different colonies.Miniprep of the colonies of Ade2::Kan-RFP (2).Digest the 3 plasmids and the neg of the 3 plasmids with PmeI (His3::Nat-RFP, Gal4::His3-RFP, Ade4::Ura3-RFP). Digest of His3::Nat-RFP, Ade4::Ura3-RFP, Gal4::His3-RFPorder (0,1,neg).Colony PCR of Ade2:KanMX-RFP (1,2,neg). Gel verification (poor results). Taylor - Re-did gel of PCR #11-GFP appears to be in all 12 colonies screened (1-12, 0, neg). 2 PCRamplifications. July 04 Emily - Good growth on plates. Inoculate 3 red colonies from each plate in 3mL LB Kan and leave togrow for 6 hours. Miniprep and nanodrop. Taylor - Redo the 2 PCR amplifications. Digest pSB1K3 and RFP (both type 2S) with Bsa1. Ligation of the two parts. E.coli transformation. July 05 Emily - Digest minipreps with EcoRI and SpeI to see if they contain the correct plasmids. Gel verification (good results) except for Gal4 2 and Ade4 3. Redo second KanMX PCR to restock in preparation. Gel verification (good results). PCR purification and nanodrop. Victoria - Colony PCR and inoculation of Ade2::Kan-RFP. Miniprep of the colonies of Ade2::Kan-RFP.PCR of the RFP plasmids and the ones without, there are 4 plasmids. Have different primers for each so that it cuts out the homologies, RFP and selection marker together. E.coli transformation. July 06 Victoria - Inoculated the plasmids with RFP for glycerol stock and more minipreps for purified DNAin 5mL LB Kan. Digested yeGFP and the 4 plasmids: Ade2:Kan-RFP, Ade4:Ura3-RFP, Gal4:His3-RFP, His3:Nat-RFP (purified product) with Not1. Ligated yeGFP and the 4 digested plasmids. Miniprepped the inoculated product and made glycerol stocks. PCR of the GFP 2A seeding and did a purification after verifying through gel (good results). July 08 Emily - Inoculate yeasts 4, 5, 6, 8, 10 (plus negative) from the URA3-GPD promoter-amilCP-CYC1 terminator patch plate in 5mL YPD. Incubate overnight at 30°C. Also patch 4, 5, 6, 8, 10 on another -Ura plate. Digest homologies-RFP-PSB1K3 plus MfeI-KanMX-EXSP. Heat shock. Ligate. E.coli transformation. Victoria - Inoculated the 4 plasmids with yeGFP. Did a colony PCR of the 4 plasmids. Gel verification (poor results). Taylor - Good growth. Inoculate colonies. Redo 3 PCR amplifications. Miniprep and nanodrop. July 09 Emily - Redo the Gal4 transformation. Heat shock. Ligate. Make glycerol stock of yesterday’s yeastinoculation (750uL 30% glycerol and 750uL inoculation). Store in iGEM 2019 box in -80°C freezer. Inoculate 9 colonies and a negative overnight from the Ade2 plate from yesterday’s transformation. Victoria - Inoculated 5 colonies for Ade2:Kan-yeGFP. Screen 5 colonies for each plates of Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP; only His3:Nat-yeGFP that has 1 colony screened. Taylor - Digested the 3 pSB1K3/RFP Type 2S tubes with Bsa1 so that verification would be done. Glycerol stocked. Inoculated colonies #3 and #11 for GFP open reading frame characterization in 10 mL YPD-put in 30 °C fridge overnight. Gel verification (poor results). July 10 Emily - Make glycerol stock of yesterday’s Ade2 KanMX inoculation and miniprep it. Lots of growth (too much) on yesterday’s transformations. Digest the minipreps with PmeI. . Gel verification (good results). Victoria - Miniprep. Taylor - Attempted to PCR verify the GFP (since the gels were not showing anything from the digest). Made glycerol stop of GFP open reading frame in yeast. Gel verification (poor results). Redo Process of creating RFP/pSB1K3 Type 2S. July 11 Victoria - Digest all plasmids of yeGFP with NotI overnight. July 12 Victoria - Gel verification (poor results), doesn’t contain yeGFP. July 15 Emily - Colony PCR. Miniprep PCR. Victoria - Throw out the digests that do not have the yeGFP minipreps and digests of the yeGFP containg plasmids. Screen 5 more colonies of Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP innoculate itovernight. Colony PCR of the 5 colonies of Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP. July 16 Emily - Colony PCR. Miniprep PCR. Victoria - Miniprep the inoculated colonies and nanodrop. Digest of minipreps with NotI. Taylor - PCR amplifications for pSB1K3 and RFP for type 2S. Gel and got no contamination in the 0 or negative lanes. PCR purification. Nanodrop. July 17 Victoria - Gel verification (poor results). July 18 Victoria - New method. Using Taylor’s GFP plasmid since it contains the insert yeGFP. Colony PCR of Taylor’s GFP amplify with qq19 and qq11 (11). Gel the colony PCR products. Purify the PCR products that contain the yeGFP. PCR amplified with pp22 and pp23. Gel verification (poor results). July 19 Victoria - Digest with different enzymes SpeI and EcoRI. Digest the plasmids with RFP then digest of the ones containing GFP. Gel verification (usable results). Purification. July 22 Victoria - Gel His3:Nat-RFP, Ade2:Kan-RFP, Ade4:Ura3-RFP, Gal4:His3-RFP and GFP (1-3). Ligation ofthe 4 backbones and the insert GFP. E.coli transformation of the ligase with different methods changing one step, the incubation time in 42C of 1.40secs versus. July 24 Victoria - The E.coli transformation has colonies in all plates. Colony PCR with pp22 and pp23 andanother set with qq37 and qq38. Inoculated 5 tubes of His3:Nat-yeGFP. Gel verification, 3 plasmids haveyeGFP - Ade2:Kan, Ade4:Ura3, Gal4:His3. His3:Gal4 that doesn’t contain yeGFP. July 25 Victoria - Colony PCR of His3:Nat-yeGFP tubes. Miniprep His3:Nat-yeGFP, Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP. Glycerol stocked all of the plasmids with yeGFP. July 26 Victoria - Digest with pme1 , the gel didn’t show the bands at the right length, thus it doesn’t contain yeGFP , only Ade4:Ura3-yeGFP might which is a mixed colony might contain the yeGFP. False positive. July 29 Victoria - Digest with pme1 for other the 5 inoculated tubes of each plasmids with possible yeGFP after a miniprep. Gel all the colonies but none had yeGFP. Inoculated to screen 3 colonies for each plasmids with possible yeGFP. July 30 Victoria - Re-gel the digests of Gal4:His3-yeGFP and His3:Nat-yeGFP, it did not show yeGFP. Miniprep the 3 inoculated tubes of each plasmids with possible yeGFP. PCR the miniprep products, zeros which are the plasmids with RFP as comparison and a negative with primers qq37 and qq38, elongation time: 38secs, annealing temperature: 64C. Tested the competent cells that Setti made; 100, 200, 500, neg of a psb1k3. Streaked glycerol stock of Ade4:Ura3-yeGFP that could possibly have yeGFP. Gel verification (poor results). July 31 Victoria - PCR with pp22 and pp23 of Ade4:Ura3-yeGFP to hopefully extract the band that contains the yeGFP. PCR with qq37 and qq38 of the yeGFP yeast of Taylor’s plasmids. Troubleshoot with DMSO and annealing temperature. Gel verification (poor results) due to too many parameters at once. August 09 Victoria - Obtained biobricks of Setti’s yeGFP and Taylor’s yeGFP insert. 2 PCR amplifications. August 10 Victoria - Gel on the 2 PCRs, yeGFP are shown at right lengths (good results). Digest the Ade2:Kan-RFP, Ade4:Ura3-RFP, Gal4:His3-RFP, His3:Nat-RFP, the 2 purified yeGFP with Ecor1 and Spe1 (Tay’s) and Not1 (Setti’s). Need to re-digest yeGFP Setti’s cassette with Not1 if it doesn’t work after plating. Completed ligation math for both Setti’s and Tay’s yeGFP cassette. August 11 Victoria - Ligation. Clear fridge. E.coli transformation. August 12 Victoria - Inoculation of 13 tubes of Ade2:Kan-yeGFP. August 13 Victoria - Miniprep 12 tubes of Ade2:Kan-yeGFP. Digest with NotI. Gel verification (poor results). August 14 Victoria - Inoculated 3 colonies for each plasmid (Ade4:Ura3-yeGFP, Gal4:His3-yeGFP, His3:Nat-yeGFP).Streak strain to plate on LB Kan for the 4 RFP plasmids. Inoculate in LB Kan of the 4 RFP plasmids (Ade4:Ura3-RFP, Gal4:His3-RFP, His3:Nat-RFP, Ade2:Kan-RFP). Digest the purified Setti’s biobrick yeGFP with Ecor1 and Spe1. August 15 Victoria - Miniprep all 4 tubes of the RFP plasmids (Ade4:Ura3-RFP, Gal4:His3-RFP, His3:Nat-RFP, Ade2:Kan-RFP) and 2 tubes of each plasmid Tay’s yeGFP (Ade4:Ura3-yeGFP, Gal4:His3-yeGFP, His3:Nat-yeGFP).Digest the plasmids of RFP with Ecor1 and Spe1 HF. Digest the plasmid of Tay’s yeGFP with Not1 HF. Gel Tay’s yeGFP (His3:Nat-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP) of 2 tubes each. August 16 Victoria - Ligation of Setti’s yeGFP and RFP plasmids. E.coli transformation and plate on LB Kan. August 17 Victoria - Inoculation of 2 tubes each for the plasmids Setti Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP and His3:Nat-yeGFP. August 18 Victoria - Miniprep all plasmids Setti Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP and His3:Nat-yeGFP. Digest miniprep with Not1 and Pme1. Verification through gel (good results, confirmed inserts!) Inoculate BY4742. Glycerol stock of the 4 plasmids. August 19 Victoria - Yeast transformation Setti Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP, Gal4:His3-yeGFP. August 21 Victoria - Replica for the plates His- and Ura- of the plasmids Gal4:His3-yeGFP and Ade4:Ura3-yeGFP with His and Ura expressed plates respectively. The ura- plate looked as if there is a graveyard. Digest pme1, gel for His3:Nat-yeGFP and Ade4:Ura3-yeGFP. Inoculate BY4742 August 22 Victoria - Yeast transformation with digests for His3:Nat-yeGFP and Ade4:Ura3-yeGFP ; on plates YPD nat and ura- August 28 Victoria - Only 3 of the plates has grown yeast : Gal4:His3-yeGFP, Ade2:Kan-yeGFP, Ade4:Ura3-yeGFP. The one that did not grow is His3:Nat-yeGFP.

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