Difference between revisions of "Team:Chalmers-Gothenburg/Schedule"
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− | {{ | + | {{Chalmers-Gothenburg}} |
− | < | + | <h2>Lab book compilation</h2> |
− | + | <h3>June</h3> | |
− | + | <p><b>9th</b><br> | |
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− | <p><b>9th | + | |
Preparation of 10x TE, 10x LiOAc, LiSorb, PEG/LiOAc and 200mM LiOAc + 1% SDS stocks</p> | Preparation of 10x TE, 10x LiOAc, LiSorb, PEG/LiOAc and 200mM LiOAc + 1% SDS stocks</p> | ||
− | <p><b>10th | + | <p><b>10th</b><br> |
Extraction of genomic DNA from Saccharomyces cerevisiae CEN PK 113 – 11C strain</p> | Extraction of genomic DNA from Saccharomyces cerevisiae CEN PK 113 – 11C strain</p> | ||
− | <p><b>17th | + | <p><b>17th</b><br> |
Inoculation of plasmids for all the constructs in Escherichia coli DH5α strain</p> | Inoculation of plasmids for all the constructs in Escherichia coli DH5α strain</p> | ||
− | <p><b>18th | + | <p><b>18th</b><br> |
Purification of plasmids using plasmid miniprep kit</p> | Purification of plasmids using plasmid miniprep kit</p> | ||
− | <p><b>25th | + | <p><b>25th</b><br> |
Received all genes for Rec proteins in G-blocks, resuspended said G-blocks in TE buffer</p> | Received all genes for Rec proteins in G-blocks, resuspended said G-blocks in TE buffer</p> | ||
− | <p><b>29th | + | <p><b>29th</b><br> |
PCR to extract all fragments, for construct 4, 5, 6, 7 and 8 (Excl. Mam2 and Rec genes) from genomic DNA of S. cerevisiae.</p> | PCR to extract all fragments, for construct 4, 5, 6, 7 and 8 (Excl. Mam2 and Rec genes) from genomic DNA of S. cerevisiae.</p> | ||
− | <p><b>30th | + | <p><b>30th</b><br> |
Inoculation of Schizosaccharomyces pombe (L972 h+ and Δ8 h- strains)<br> | Inoculation of Schizosaccharomyces pombe (L972 h+ and Δ8 h- strains)<br> | ||
Extraction of genomic DNA from S. pombe L972 h+ strain<br> | Extraction of genomic DNA from S. pombe L972 h+ strain<br> | ||
Line 345: | Line 22: | ||
Redone PCR of failed PCR products (TDH, PGK), also added Mam2 to PCR</p> | Redone PCR of failed PCR products (TDH, PGK), also added Mam2 to PCR</p> | ||
− | < | + | <h3>July</h3> |
− | <p><b>1st | + | <p><b>1st</b><br> |
Gel electrophoresis on PCR (TDH, PGK and Mam2)<br> | Gel electrophoresis on PCR (TDH, PGK and Mam2)<br> | ||
Nanodrop of purified PCR products <br> | Nanodrop of purified PCR products <br> | ||
Line 353: | Line 30: | ||
PCR purification of Mam2 using PCR purification miniprep kit<br> | PCR purification of Mam2 using PCR purification miniprep kit<br> | ||
New PCR run on TDH and PGK</p> | New PCR run on TDH and PGK</p> | ||
− | <p><b>2nd | + | <p><b>2nd</b><br> |
Gel electrophoresis of TDH and PGK <br> | Gel electrophoresis of TDH and PGK <br> | ||
Gibson assembly on construct 5<br> | Gibson assembly on construct 5<br> | ||
Line 359: | Line 36: | ||
Gel electrophoresis on PCR products (TDH)<br> | Gel electrophoresis on PCR products (TDH)<br> | ||
Transformation of construct 5 into E. coli and incubation on plate for 16 hours</p> | Transformation of construct 5 into E. coli and incubation on plate for 16 hours</p> | ||
− | <p><b>3rd | + | <p><b>3rd</b><br> |
Gel electrophoresis on TDH using GelGreen<br> | Gel electrophoresis on TDH using GelGreen<br> | ||
PCR on TDH<br> | PCR on TDH<br> | ||
Line 365: | Line 42: | ||
Took colonies from E. coli containing construct 5 for colony PCR and new plate<br> | Took colonies from E. coli containing construct 5 for colony PCR and new plate<br> | ||
Gel electrophoresis on colony PCR</p> | Gel electrophoresis on colony PCR</p> | ||
− | <p><b>5th | + | <p><b>5th</b><br> |
Inoculation of colony 5 from colony PCR</p> | Inoculation of colony 5 from colony PCR</p> | ||
− | <p><b>6th | + | <p><b>6th</b><br> |
Purification of PCR products (TDH) <br> | Purification of PCR products (TDH) <br> | ||
Linearization of plasmid (p0255-pX-2-URA)<br> | Linearization of plasmid (p0255-pX-2-URA)<br> | ||
Line 375: | Line 52: | ||
Gibson assembly of RecJ, RecD2 and RecQ (Split on delivery in G-blocks)<br> | Gibson assembly of RecJ, RecD2 and RecQ (Split on delivery in G-blocks)<br> | ||
Inoculation of S. cerevisiae CEN.PK2</p> | Inoculation of S. cerevisiae CEN.PK2</p> | ||
− | <p><b>7th | + | <p><b>7th</b><br> |
Gibson assembly of construct 4 <br> | Gibson assembly of construct 4 <br> | ||
Transformation of construct 4 into E. coli and incubation on plate for 16 hours<br> | Transformation of construct 4 into E. coli and incubation on plate for 16 hours<br> | ||
Line 384: | Line 61: | ||
PCR to amplify PprA, RecD2, RecJ, RecQ<br> | PCR to amplify PprA, RecD2, RecJ, RecQ<br> | ||
Gel electrophoresis on PCR products (PprA, RecD2, RecJ, RecQ)</p> | Gel electrophoresis on PCR products (PprA, RecD2, RecJ, RecQ)</p> | ||
− | <p><b>8th | + | <p><b>8th</b><br> |
PCR on pCYC1m using PCR products as template<br> | PCR on pCYC1m using PCR products as template<br> | ||
Gel electrophoresis on PCR products (pCYC1m)<br> | Gel electrophoresis on PCR products (pCYC1m)<br> | ||
Line 395: | Line 72: | ||
PCR to amplify PprA, RecD2, RecJ, RecQ<br> | PCR to amplify PprA, RecD2, RecJ, RecQ<br> | ||
Gel on PCR products (PprA)</p> | Gel on PCR products (PprA)</p> | ||
− | <p><b>9th | + | <p><b>9th</b><br> |
Gel electrophoresis on Colony PCR (construct 4) <br> | Gel electrophoresis on Colony PCR (construct 4) <br> | ||
Inoculation of S. pombe<br> | Inoculation of S. pombe<br> | ||
Line 407: | Line 84: | ||
Gel electrophoresis on RecJ, RecQ, RecD2<br> | Gel electrophoresis on RecJ, RecQ, RecD2<br> | ||
Gel electrophoresis on PCR products (RecJ RecQ RecD2) using GelGreen</p> | Gel electrophoresis on PCR products (RecJ RecQ RecD2) using GelGreen</p> | ||
− | <p><b>10th | + | <p><b>10th</b><br> |
PCR of dCas9/Vp64 using three different available templates<br> | PCR of dCas9/Vp64 using three different available templates<br> | ||
Gibson assembly of construct 1<br> | Gibson assembly of construct 1<br> | ||
Line 415: | Line 92: | ||
PCR on RecA and Ssb<br> | PCR on RecA and Ssb<br> | ||
Gel electrophoresis on PCR products (RecA and Ssb)</p> | Gel electrophoresis on PCR products (RecA and Ssb)</p> | ||
− | <p><b>13th | + | <p><b>13th</b><br> |
Colony PCR of construct 4<br> | Colony PCR of construct 4<br> | ||
Transformation of construct 1 into E.coli and incubation on plate for 16 hours<br> | Transformation of construct 1 into E.coli and incubation on plate for 16 hours<br> | ||
Line 421: | Line 98: | ||
Linearization of plasmid (p0389) using AsisI<br> | Linearization of plasmid (p0389) using AsisI<br> | ||
Gibson assembly of construct 6, 7 and 8</p> | Gibson assembly of construct 6, 7 and 8</p> | ||
− | <p><b>14th | + | <p><b>14th</b><br> |
Gel electrophoresis on Colony PCR (construct 4)<br> | Gel electrophoresis on Colony PCR (construct 4)<br> | ||
PCR on dCas9/Vp64 using PrimeStar polymerase (Previously Phusion HF)<br> | PCR on dCas9/Vp64 using PrimeStar polymerase (Previously Phusion HF)<br> | ||
Line 429: | Line 106: | ||
Transformation of constructs 6, 7 and 8 into E .coli and incubation on plate for 16 hours<br> | Transformation of constructs 6, 7 and 8 into E .coli and incubation on plate for 16 hours<br> | ||
Preparation of –LEU YPD plates</p> | Preparation of –LEU YPD plates</p> | ||
− | <p><b>15th | + | <p><b>15th</b><br> |
Linearization of plasmid (p0255 pX-2-URA) using AsiSI<br> | Linearization of plasmid (p0255 pX-2-URA) using AsiSI<br> | ||
PCR on pCYC1m and pFUS1<br> | PCR on pCYC1m and pFUS1<br> | ||
Line 436: | Line 113: | ||
Linearization of plasmids using AsiSI and NotI<br> | Linearization of plasmids using AsiSI and NotI<br> | ||
Transformation and Gel electrophoresis of negative control for construct 6, 7 and 8</p> | Transformation and Gel electrophoresis of negative control for construct 6, 7 and 8</p> | ||
− | <p><b>16th | + | <p><b>16th</b><br> |
PCR on dCas9/Vp64<br> | PCR on dCas9/Vp64<br> | ||
Gel purification of dCas9/Vp64<br> | Gel purification of dCas9/Vp64<br> | ||
Linearization of plasmids (p0255, p0389) using NotI<br> | Linearization of plasmids (p0255, p0389) using NotI<br> | ||
Positive control of –LEU YPD plates</p> | Positive control of –LEU YPD plates</p> | ||
− | <p><b>17th | + | <p><b>17th</b><br> |
Gel electrophoresis on PCR products (dCas9/Vp64) using GelGreen <br> | Gel electrophoresis on PCR products (dCas9/Vp64) using GelGreen <br> | ||
Concentration of extracted products (dCas9/Vp64)<br> | Concentration of extracted products (dCas9/Vp64)<br> | ||
Preparation of –HIS plates<br> | Preparation of –HIS plates<br> | ||
Verification of linearization of construct 5 plasmid using NotI</p> | Verification of linearization of construct 5 plasmid using NotI</p> | ||
− | <p><b>20th | + | <p><b>20th</b><br> |
Verification of linearization of construct 5 plasmid using NotI</p> | Verification of linearization of construct 5 plasmid using NotI</p> | ||
− | <p><b>21st | + | <p><b>21st</b><br> |
Restriction of p0259 using NotI for positive control<br> | Restriction of p0259 using NotI for positive control<br> | ||
Inoculation of S. cerevisiae CEN.PK2</p> | Inoculation of S. cerevisiae CEN.PK2</p> | ||
− | <p><b>22nd | + | <p><b>22nd</b><br> |
PCR purification of 2-pFUS1 and 3-pCYC1m<br> | PCR purification of 2-pFUS1 and 3-pCYC1m<br> | ||
Transformation of construct 5 into yeast <br> | Transformation of construct 5 into yeast <br> | ||
Inoculation of plasmids (p0257, p0258, p0388, p0391) in E.coli<br> | Inoculation of plasmids (p0257, p0258, p0388, p0391) in E.coli<br> | ||
Plasmid linearization for constructs 1/4, 2, 3, 6, 7 and 8 using AsiSI</p> | Plasmid linearization for constructs 1/4, 2, 3, 6, 7 and 8 using AsiSI</p> | ||
− | <p><b>23rd | + | <p><b>23rd</b><br> |
Purification of plasmids using miniprep<br> | Purification of plasmids using miniprep<br> | ||
Inoculation of plasmids (p0255 and p0389) in E.coli <br> | Inoculation of plasmids (p0255 and p0389) in E.coli <br> | ||
Plasmid linearization for constructs 2, 3, 7, 8 using AsiSI<br> | Plasmid linearization for constructs 2, 3, 7, 8 using AsiSI<br> | ||
Gel electrophoresis on linearized plasmids (constructs 2, 3, 7, 8) using GelGreen </p> | Gel electrophoresis on linearized plasmids (constructs 2, 3, 7, 8) using GelGreen </p> | ||
− | <p><b>24th | + | <p><b>24th</b><br> |
Concentration of linearized plasmids p0257, p0258, p0388, p0389<br> | Concentration of linearized plasmids p0257, p0258, p0388, p0389<br> | ||
Gibson assembly of construct 7 and 8</p> | Gibson assembly of construct 7 and 8</p> | ||
− | <p><b>27th | + | <p><b>27th</b><br> |
Purification and linearization of plasmids p0255, p0389<br> | Purification and linearization of plasmids p0255, p0389<br> | ||
− | Transformation of constructs 7 and 8 into E .coli and incubation on plate for 16 hours< | + | Transformation of constructs 7 and 8 into E .coli and incubation on plate for 16 hours</p> |
− | <p><b>28th | + | <p><b>28th</b><br> |
Colony PCR of construct 7<br> | Colony PCR of construct 7<br> | ||
Transformation of construct 8 into E. coli<br> | Transformation of construct 8 into E. coli<br> | ||
Linearization of construct 5 (p0259) using NotI<br> | Linearization of construct 5 (p0259) using NotI<br> | ||
Inoculation of construct 5 in yeast CEN.PK2</p> | Inoculation of construct 5 in yeast CEN.PK2</p> | ||
− | <p><b>29th | + | <p><b>29th</b><br> |
Gel electrophoresis on colony PCR of construct 7<br> | Gel electrophoresis on colony PCR of construct 7<br> | ||
Transformation of construct 5 into yeast CEN.PK2<br> | Transformation of construct 5 into yeast CEN.PK2<br> | ||
Line 478: | Line 155: | ||
Transformation of construct 4 into E. coli<br> | Transformation of construct 4 into E. coli<br> | ||
Plating of construct 4 in E. coli</p> | Plating of construct 4 in E. coli</p> | ||
− | <p><b>30th | + | <p><b>30th</b><br> |
Plating of construct 4 in E. coli<br> | Plating of construct 4 in E. coli<br> | ||
Colony PCR on construct 4 in p0257<br> | Colony PCR on construct 4 in p0257<br> | ||
Gel electrophoresis on construct 4<br> | Gel electrophoresis on construct 4<br> | ||
Suspension of BioBrick primers</p> | Suspension of BioBrick primers</p> | ||
− | <p><b>31st | + | <p><b>31st</b><br> |
Verification of PUR parts<br> | Verification of PUR parts<br> | ||
PCR on BioBricks<br> | PCR on BioBricks<br> | ||
Line 490: | Line 167: | ||
PCR on backbone with primeSTAR</p> | PCR on backbone with primeSTAR</p> | ||
− | < | + | <h3>August</h3> |
− | <p><b>3rd | + | <p><b>3rd</b><br> |
Inoculation of construct 4 in E. coli<br> | Inoculation of construct 4 in E. coli<br> | ||
Gel electrophoresis on RecJ using GelGreen <br> | Gel electrophoresis on RecJ using GelGreen <br> | ||
Line 497: | Line 174: | ||
Transformation of construct 5 into yeast<br> | Transformation of construct 5 into yeast<br> | ||
PCR purification of plasmids</p> | PCR purification of plasmids</p> | ||
− | <p><b>4th | + | <p><b>4th</b><br> |
Purification of plasmid containing construct 4 using plasmid miniprep kit<br> | Purification of plasmid containing construct 4 using plasmid miniprep kit<br> | ||
Gibson assembly of construct 1, 2, 7 and 8<br> | Gibson assembly of construct 1, 2, 7 and 8<br> | ||
Line 506: | Line 183: | ||
Restriction prior yeast transform<br> | Restriction prior yeast transform<br> | ||
PCR purification of BioBricks RecA, PprA, Ssb, SteMam</p> | PCR purification of BioBricks RecA, PprA, Ssb, SteMam</p> | ||
− | <p><b>5th | + | <p><b>5th</b><br> |
2nd PCR of BioBricks RecA, PprA, Ssb, SteMam<br> | 2nd PCR of BioBricks RecA, PprA, Ssb, SteMam<br> | ||
Gel electrophoresis on 2nd PCR of BioBricks<br> | Gel electrophoresis on 2nd PCR of BioBricks<br> | ||
Line 513: | Line 190: | ||
Inoculation of S. cerevisiae INFD-70 strain<br> | Inoculation of S. cerevisiae INFD-70 strain<br> | ||
Inoculation of E. coli</p> | Inoculation of E. coli</p> | ||
− | <p><b>6th | + | <p><b>6th</b><br> |
Purification of plasmid (p425) using plasmid miniprep kit<br> | Purification of plasmid (p425) using plasmid miniprep kit<br> | ||
PCR of construct 5 from gDNA<br> | PCR of construct 5 from gDNA<br> | ||
Line 520: | Line 197: | ||
Linearization of construct 4<br> | Linearization of construct 4<br> | ||
Preparation of 1M sorbitol</p> | Preparation of 1M sorbitol</p> | ||
− | <p><b>7th | + | <p><b>7th</b><br> |
Gel extraction of RecJ<br> | Gel extraction of RecJ<br> | ||
Growing of S. pombe<br> | Growing of S. pombe<br> | ||
Line 529: | Line 206: | ||
PCR purification of RecQ, RecD2, RecJ, RecA, PprA, Ssb<br> | PCR purification of RecQ, RecD2, RecJ, RecA, PprA, Ssb<br> | ||
Gel electrophoresis on RecJ</p> | Gel electrophoresis on RecJ</p> | ||
− | <p><b>10th | + | <p><b>10th</b><br> |
Growing of S. pombe<br> | Growing of S. pombe<br> | ||
Replated inoculation of construct 1, 2 and 8<br> | Replated inoculation of construct 1, 2 and 8<br> | ||
Line 537: | Line 214: | ||
Transformation of BioBrick vector into E.coli<br> | Transformation of BioBrick vector into E.coli<br> | ||
Preparation of media for Safety Switch testing</p> | Preparation of media for Safety Switch testing</p> | ||
− | <p><b>11th | + | <p><b>11th</b><br> |
PCR on RecJ | PCR on RecJ | ||
Purification of construct 1, 2 and 8 using miniprep<br> | Purification of construct 1, 2 and 8 using miniprep<br> | ||
Line 543: | Line 220: | ||
Restriction of plasmids for construct 1, 2 and 8<br> | Restriction of plasmids for construct 1, 2 and 8<br> | ||
Verification of construct 2 and 8</p> | Verification of construct 2 and 8</p> | ||
− | <p><b>12th | + | <p><b>12th</b><br> |
Gel extraction of RecJ<br> | Gel extraction of RecJ<br> | ||
Preparation of chloramphenicol plates<br> | Preparation of chloramphenicol plates<br> | ||
Testing Safety Switch<br> | Testing Safety Switch<br> | ||
PCR using PrimeSTAR p0391 (8)</p> | PCR using PrimeSTAR p0391 (8)</p> | ||
− | <p><b>13th | + | <p><b>13th</b><br> |
OD measurements of Safety Switch and wildtype<br> | OD measurements of Safety Switch and wildtype<br> | ||
Gel electrophoresis on PCR <br> | Gel electrophoresis on PCR <br> | ||
Line 555: | Line 232: | ||
Transformation of pSB1C3 into E .coli<br> | Transformation of pSB1C3 into E .coli<br> | ||
Calculations on Safety Switch test</p> | Calculations on Safety Switch test</p> | ||
− | <p><b>14th | + | <p><b>14th</b><br> |
Gel electrophoresis on PUR samples<br> | Gel electrophoresis on PUR samples<br> | ||
Gibson assembly of construct 6, 7 and 8<br> | Gibson assembly of construct 6, 7 and 8<br> | ||
Line 564: | Line 241: | ||
Restriction of BioBrick vector<br> | Restriction of BioBrick vector<br> | ||
Continued test Safety Switch in YPG</p> | Continued test Safety Switch in YPG</p> | ||
− | <p><b>18th | + | <p><b>18th</b><br> |
Ligation of BioBricks<br> | Ligation of BioBricks<br> | ||
Transformation of BioBrick and construct 6, 7 and 8 into E.coli<br> | Transformation of BioBrick and construct 6, 7 and 8 into E.coli<br> | ||
Restriction of BioBrick vector</p> | Restriction of BioBrick vector</p> | ||
− | <p><b>19th | + | <p><b>19th</b><br> |
Ligation of BioBricks<br> | Ligation of BioBricks<br> | ||
Gel electrophoresis on construct 6, 7 and 8<br> | Gel electrophoresis on construct 6, 7 and 8<br> | ||
Line 575: | Line 252: | ||
Ligation of BioBrick TPI and CGC-CAS <br> | Ligation of BioBrick TPI and CGC-CAS <br> | ||
Transformation of BioBrick TPI and CGC-CAS into E. coli</p> | Transformation of BioBrick TPI and CGC-CAS into E. coli</p> | ||
− | <p><b>20th | + | <p><b>20th</b><br> |
Purification of construct 6 and 8<br> | Purification of construct 6 and 8<br> | ||
Transformation of BioBrick into E. coli</p> | Transformation of BioBrick into E. coli</p> | ||
− | <p><b>24th | + | <p><b>24th</b><br> |
Restriction of construct 6 and 8<br> | Restriction of construct 6 and 8<br> | ||
Gel electrophoresis on construct 6 and 8<br> | Gel electrophoresis on construct 6 and 8<br> | ||
PCR of Safety Switch with RFP and BioBrick Safety Switch<br> | PCR of Safety Switch with RFP and BioBrick Safety Switch<br> | ||
Filtrating S. pombe</p> | Filtrating S. pombe</p> | ||
− | <p><b>25th | + | <p><b>25th</b><br> |
Purification of plasmid containing BioBricks (RecA, Ste, Mam, SteMam)<br> | Purification of plasmid containing BioBricks (RecA, Ste, Mam, SteMam)<br> | ||
Restriction of BioBricks inserts (RecA, Ste, Mam, SteMam)<br> | Restriction of BioBricks inserts (RecA, Ste, Mam, SteMam)<br> | ||
Line 590: | Line 267: | ||
Restriction and ligation of BioBrick TEF and SUC<br> | Restriction and ligation of BioBrick TEF and SUC<br> | ||
Gibson assembly of Safety Switch with RFP</p> | Gibson assembly of Safety Switch with RFP</p> | ||
− | <p><b>26th | + | <p><b>26th</b><br> |
Gel electrophoresis of restriction fragments for BioBricks TEF and SUC<br> | Gel electrophoresis of restriction fragments for BioBricks TEF and SUC<br> | ||
Transformation of BioBrick TEFSUC, SUC RFP, TEFSUC RFP into E. coli<br> | Transformation of BioBrick TEFSUC, SUC RFP, TEFSUC RFP into E. coli<br> | ||
Restriction and ligation of BioBricks<br> | Restriction and ligation of BioBricks<br> | ||
Sequencing of parts</p> | Sequencing of parts</p> | ||
− | <p><b>27th | + | <p><b>27th</b><br> |
3rd transformation of construct 4 into yeast<br> | 3rd transformation of construct 4 into yeast<br> | ||
Gibson assembly of construct 6, 7 and 8 without vector<br> | Gibson assembly of construct 6, 7 and 8 without vector<br> | ||
Line 602: | Line 279: | ||
Gibson assembly of TEFSUC RFP and SUC RFP<br> | Gibson assembly of TEFSUC RFP and SUC RFP<br> | ||
Purification of BioBrick TEFSUC plasmid</p> | Purification of BioBrick TEFSUC plasmid</p> | ||
− | <p><b>28th | + | <p><b>28th</b><br> |
Restriction of construct 4 prior to transformation<br> | Restriction of construct 4 prior to transformation<br> | ||
2nd transformation of Safety Switch RFP to E. coli<br> | 2nd transformation of Safety Switch RFP to E. coli<br> | ||
Line 609: | Line 286: | ||
Restriction of plasmid containing BioBrick TEFSUC1<br> | Restriction of plasmid containing BioBrick TEFSUC1<br> | ||
Gel of plasmid containing BioBrick TEFSUC1 digested with PstI, PstI + EcoRI</p> | Gel of plasmid containing BioBrick TEFSUC1 digested with PstI, PstI + EcoRI</p> | ||
− | <p><b>30th | + | <p><b>30th</b><br> |
Inoculation of construct 4 in CEN.PK2<br> | Inoculation of construct 4 in CEN.PK2<br> | ||
Inoculation and re-plating of BioBrick TPI, SteMam, RecA, PprA and Ssb</p> | Inoculation and re-plating of BioBrick TPI, SteMam, RecA, PprA and Ssb</p> | ||
− | <p><b>31st | + | <p><b>31st</b><br> |
Purification of plasmids containing BioBrick TPI, SteMam, RecA, PprA and Ssb<br> | Purification of plasmids containing BioBrick TPI, SteMam, RecA, PprA and Ssb<br> | ||
Restriction of BioBrick TPI, SteMam, RecA, PprA and Ssb<br> | Restriction of BioBrick TPI, SteMam, RecA, PprA and Ssb<br> | ||
Line 619: | Line 296: | ||
Inoculation and plating of construct 4 in CEN.PK2</p> | Inoculation and plating of construct 4 in CEN.PK2</p> | ||
− | < | + | <h3>September</h3> |
− | <p><b>1st | + | <p><b>1st</b><br> |
Genomic DNA extraction of construct 4 in CEN.PK2<br> | Genomic DNA extraction of construct 4 in CEN.PK2<br> | ||
PCR of construct 4 from CEN.PK2 gDNA<br> | PCR of construct 4 from CEN.PK2 gDNA<br> | ||
Line 629: | Line 306: | ||
Gel electrophoresis on Safety Switch with RFP<br> | Gel electrophoresis on Safety Switch with RFP<br> | ||
Restriction of Safety Switch with RFP</p> | Restriction of Safety Switch with RFP</p> | ||
− | <p><b>2nd | + | <p><b>2nd</b><br> |
Gel electrophoresis of construct 4 <br> | Gel electrophoresis of construct 4 <br> | ||
1st test of construct 4<br> | 1st test of construct 4<br> | ||
Line 635: | Line 312: | ||
Restriction of plasmid prior to RFP transformation<br> | Restriction of plasmid prior to RFP transformation<br> | ||
Transformation of BioBrick TEFSUC into E. coli</p> | Transformation of BioBrick TEFSUC into E. coli</p> | ||
− | <p><b>3rd | + | <p><b>3rd</b><br> |
Concentration of pheromones<br> | Concentration of pheromones<br> | ||
Overnight cultivation of CEN.PK2 with m- and p-factor pheromones from S. pombe<br> | Overnight cultivation of CEN.PK2 with m- and p-factor pheromones from S. pombe<br> | ||
Inoculation of construct 4 in E. coli<br> | Inoculation of construct 4 in E. coli<br> | ||
OD measurements for Safety Switch TPK2</p> | OD measurements for Safety Switch TPK2</p> | ||
− | <p><b>4th | + | <p><b>4th</b><br> |
2nd test of Safety Switch TPK2 and WT<br> | 2nd test of Safety Switch TPK2 and WT<br> | ||
Restriction of BioBrick vector and TEFSUC<br> | Restriction of BioBrick vector and TEFSUC<br> | ||
Ligation of BioBrick TEFSUC with BioBrick vector</p> | Ligation of BioBrick TEFSUC with BioBrick vector</p> | ||
− | <p><b>7th | + | <p><b>7th</b><br> |
Preculture drop test Safety Switch TPK2<br> | Preculture drop test Safety Switch TPK2<br> | ||
Re-plating Safety Switch RFP</p> | Re-plating Safety Switch RFP</p> | ||
− | <p><b>8th | + | <p><b>8th</b><br> |
Cultivation of cells for spectrometry<br> | Cultivation of cells for spectrometry<br> | ||
PCR of BioBrick TEFSUC and Vector J04450<br> | PCR of BioBrick TEFSUC and Vector J04450<br> | ||
Line 654: | Line 331: | ||
Nanodrop of BioBrick TEFSUC and BioBrick vector<br> | Nanodrop of BioBrick TEFSUC and BioBrick vector<br> | ||
Restriction of BioBrick TEFSUC and BioBrick vector</p> | Restriction of BioBrick TEFSUC and BioBrick vector</p> | ||
− | <p><b>9th | + | <p><b>9th</b><br> |
2nd test of construct 4 <br> | 2nd test of construct 4 <br> | ||
Testing BioBricks<br> | Testing BioBricks<br> | ||
Line 663: | Line 340: | ||
Restriction and ligation of BioBrick TEFSUC<br> | Restriction and ligation of BioBrick TEFSUC<br> | ||
Transformation of BioBrick TEFSUC into E. coli</p> | Transformation of BioBrick TEFSUC into E. coli</p> | ||
− | <p><b>10th | + | <p><b>10th</b><br> |
Restriction of BioBrick and vector<br> | Restriction of BioBrick and vector<br> | ||
Purification of BioBrick and vector</p> | Purification of BioBrick and vector</p> | ||
− | <p><b>11th | + | <p><b>11th</b><br> |
Concentration of p-factor pheromone<br> | Concentration of p-factor pheromone<br> | ||
1st droptest on Safety Switch TPK<br> | 1st droptest on Safety Switch TPK<br> | ||
Line 672: | Line 349: | ||
Inoculation of WT, CEN.PK2, Safety Switch TPK2-Col 1, TEFSUC RFP, SUC RFP and RFP<br> | Inoculation of WT, CEN.PK2, Safety Switch TPK2-Col 1, TEFSUC RFP, SUC RFP and RFP<br> | ||
Re-plating and inoculation of E. coli with BioBrick TEFSUC</p> | Re-plating and inoculation of E. coli with BioBrick TEFSUC</p> | ||
− | <p><b>12th | + | <p><b>12th</b><br> |
− | 3rd test of construct 4</p> | + | 3rd test of construct 4<br> |
+ | Replating of E. coli with BioBrick vector p</p> | ||
+ | <p><b>14th</b><br> | ||
+ | Miniprep of E. coli with BioBrick vector from plate<br> | ||
+ | Restriction of product from miniprep (BioBrick vector)<br> | ||
+ | Restriction of BioBrick parts<br> | ||
+ | Gel electrophoresis on cut vector<br> | ||
+ | Ligation of BioBricks in BioBrick vector<br> | ||
+ | Transformation of BioBricks into E. coli and let to grow on plate<br> | ||
+ | Preparation of chloramphenicol plates</p> | ||
+ | <p><b>16th</b><br> | ||
+ | Inoculation of E.coli colonies containing BioBricks</p> | ||
+ | <p><b>17th</b><br> | ||
+ | Purification of BioBrick using plasmid miniprep kit<br> | ||
+ | Restriction of BioBricks<br> | ||
+ | Gel electrophoresis on BioBricks</p> | ||
+ | <p><b>18th</b><br> | ||
+ | Drying of successful BioBricks for submission to iGEM</p> | ||
+ | <html> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> |
Latest revision as of 01:33, 19 September 2015
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Lab book compilation
June
9th
Preparation of 10x TE, 10x LiOAc, LiSorb, PEG/LiOAc and 200mM LiOAc + 1% SDS stocks
10th
Extraction of genomic DNA from Saccharomyces cerevisiae CEN PK 113 – 11C strain
17th
Inoculation of plasmids for all the constructs in Escherichia coli DH5α strain
18th
Purification of plasmids using plasmid miniprep kit
25th
Received all genes for Rec proteins in G-blocks, resuspended said G-blocks in TE buffer
29th
PCR to extract all fragments, for construct 4, 5, 6, 7 and 8 (Excl. Mam2 and Rec genes) from genomic DNA of S. cerevisiae.
30th
Inoculation of Schizosaccharomyces pombe (L972 h+ and Δ8 h- strains)
Extraction of genomic DNA from S. pombe L972 h+ strain
Gel electrophoresis on all PCR products
PCR purification of PCR products using PCR purification miniprep kit
Redone PCR of failed PCR products (TDH, PGK), also added Mam2 to PCR
July
1st
Gel electrophoresis on PCR (TDH, PGK and Mam2)
Nanodrop of purified PCR products
Preparation of S. pombe for cryo-storage
Linearization of plasmid (p0259 pXII-1-LEU) for construct 5
PCR purification of Mam2 using PCR purification miniprep kit
New PCR run on TDH and PGK
2nd
Gel electrophoresis of TDH and PGK
Gibson assembly on construct 5
PCR of TDH using both genomic DNA and PCR products (TDH) as template
Gel electrophoresis on PCR products (TDH)
Transformation of construct 5 into E. coli and incubation on plate for 16 hours
3rd
Gel electrophoresis on TDH using GelGreen
PCR on TDH
Gel electrophoresis on PCR products (TDH)
Took colonies from E. coli containing construct 5 for colony PCR and new plate
Gel electrophoresis on colony PCR
5th
Inoculation of colony 5 from colony PCR
6th
Purification of PCR products (TDH)
Linearization of plasmid (p0255-pX-2-URA)
Purification of plasmid containing construct 5 using plasmid miniprep kit
PCR on plasmid with construct 5
Gel electrophoresis on PCR products (construct 5)
Gibson assembly of RecJ, RecD2 and RecQ (Split on delivery in G-blocks)
Inoculation of S. cerevisiae CEN.PK2
7th
Gibson assembly of construct 4
Transformation of construct 4 into E. coli and incubation on plate for 16 hours
PCR on dCas9/Vp64, pCYC1m, and CGC1-Cas9 genes
Gel electrophoresis on PCR products (dCas9/Vp64, pCYC1m, and CGC1)
Gel electrophoresis for verification of construct 5
Purification of PCR products (construct 5)
PCR to amplify PprA, RecD2, RecJ, RecQ
Gel electrophoresis on PCR products (PprA, RecD2, RecJ, RecQ)
8th
PCR on pCYC1m using PCR products as template
Gel electrophoresis on PCR products (pCYC1m)
Purification of PCR products (CGC1) using miniprep
Took colonies from E. coli containing construct 4 for colony PCR and new plate
Restriction verification of plasmid with construct 5 using EcoRI and NotI
Linearization of plasmid with construct 5 using NotI
Preparation of –LEU YPD plates
Inoculation of plasmids (p0257 X-3-LEU, p0258 X-4-HIS, p0388 XI-1-LEU, p0391 XI-5-HIS) in E. coli
PCR to amplify PprA, RecD2, RecJ, RecQ
Gel on PCR products (PprA)
9th
Gel electrophoresis on Colony PCR (construct 4)
Inoculation of S. pombe
PCR on dCas9/Vp64
Gel electrophoresis on PCR products (dCas9/Vp64)
Colony PCR on construct 4 colonies 2, 3, 4, 10
Gel electrophoresis on colony PCR (construct 4) and PCR products (pCYC1m)
Gel electrophoresis on PCR products (pCYC1m) using GelGreen
Purification of plasmids (p0257, p0258, p0388, p0391) from E. coli
Gel electrophoresis on PCR products (PprA) using GelGreen
Gel electrophoresis on RecJ, RecQ, RecD2
Gel electrophoresis on PCR products (RecJ RecQ RecD2) using GelGreen
10th
PCR of dCas9/Vp64 using three different available templates
Gibson assembly of construct 1
Gel electrophoresis on Colony PCR (construct 4 centrifuged since yesterday)
Gel electrophoresis on PCR products (dCas9/Vp64) and construct 4
QuickYeast transformation of construct 5 into yeast
PCR on RecA and Ssb
Gel electrophoresis on PCR products (RecA and Ssb)
13th
Colony PCR of construct 4
Transformation of construct 1 into E.coli and incubation on plate for 16 hours
QuickYeast transformation of construct 5 into yeast
Linearization of plasmid (p0389) using AsisI
Gibson assembly of construct 6, 7 and 8
14th
Gel electrophoresis on Colony PCR (construct 4)
PCR on dCas9/Vp64 using PrimeStar polymerase (Previously Phusion HF)
Gel electrophoresis on dCas9/Vp64
Transformation of linearized plasmid (p0255) into E.coli for negative control of construct 1
QuickYeast transformation of construct 5 into yeast
Transformation of constructs 6, 7 and 8 into E .coli and incubation on plate for 16 hours
Preparation of –LEU YPD plates
15th
Linearization of plasmid (p0255 pX-2-URA) using AsiSI
PCR on pCYC1m and pFUS1
Gel purification of PCR products (dCas9/Vp64)
Gel electrophoresis on PCR products (pCYC1m and pFUS1) and linearized plasmid (p0255)
Linearization of plasmids using AsiSI and NotI
Transformation and Gel electrophoresis of negative control for construct 6, 7 and 8
16th
PCR on dCas9/Vp64
Gel purification of dCas9/Vp64
Linearization of plasmids (p0255, p0389) using NotI
Positive control of –LEU YPD plates
17th
Gel electrophoresis on PCR products (dCas9/Vp64) using GelGreen
Concentration of extracted products (dCas9/Vp64)
Preparation of –HIS plates
Verification of linearization of construct 5 plasmid using NotI
20th
Verification of linearization of construct 5 plasmid using NotI
21st
Restriction of p0259 using NotI for positive control
Inoculation of S. cerevisiae CEN.PK2
22nd
PCR purification of 2-pFUS1 and 3-pCYC1m
Transformation of construct 5 into yeast
Inoculation of plasmids (p0257, p0258, p0388, p0391) in E.coli
Plasmid linearization for constructs 1/4, 2, 3, 6, 7 and 8 using AsiSI
23rd
Purification of plasmids using miniprep
Inoculation of plasmids (p0255 and p0389) in E.coli
Plasmid linearization for constructs 2, 3, 7, 8 using AsiSI
Gel electrophoresis on linearized plasmids (constructs 2, 3, 7, 8) using GelGreen
24th
Concentration of linearized plasmids p0257, p0258, p0388, p0389
Gibson assembly of construct 7 and 8
27th
Purification and linearization of plasmids p0255, p0389
Transformation of constructs 7 and 8 into E .coli and incubation on plate for 16 hours
28th
Colony PCR of construct 7
Transformation of construct 8 into E. coli
Linearization of construct 5 (p0259) using NotI
Inoculation of construct 5 in yeast CEN.PK2
29th
Gel electrophoresis on colony PCR of construct 7
Transformation of construct 5 into yeast CEN.PK2
Gibson assembly of construct 4
Transformation of construct 4 into E. coli
Plating of construct 4 in E. coli
30th
Plating of construct 4 in E. coli
Colony PCR on construct 4 in p0257
Gel electrophoresis on construct 4
Suspension of BioBrick primers
31st
Verification of PUR parts
PCR on BioBricks
Gel electrophoresis on BioBricks
2nd Gel electrophoresis on BioBricks
PCR on backbone with primeSTAR
August
3rd
Inoculation of construct 4 in E. coli
Gel electrophoresis on RecJ using GelGreen
Gel electrophoresis on backbone and BioBricks
Transformation of construct 5 into yeast
PCR purification of plasmids
4th
Purification of plasmid containing construct 4 using plasmid miniprep kit
Gibson assembly of construct 1, 2, 7 and 8
Gel electrophoresis on products of Gibson assembly
Linearization of construct 4
Gel electrophoresis on restricted construct 4
Transformation of TEF in p425 into E. coli
Restriction prior yeast transform
PCR purification of BioBricks RecA, PprA, Ssb, SteMam
5th
2nd PCR of BioBricks RecA, PprA, Ssb, SteMam
Gel electrophoresis on 2nd PCR of BioBricks
Gel electrophoresis on construct 1, 2, 7 and 8
Transformation of construct 1, 2, 7 and 8 into E. coli
Inoculation of S. cerevisiae INFD-70 strain
Inoculation of E. coli
6th
Purification of plasmid (p425) using plasmid miniprep kit
PCR of construct 5 from gDNA
Gel electrophoresis on RecJ using GelGreen
Transformation of construct 1 into yeast
Linearization of construct 4
Preparation of 1M sorbitol
7th
Gel extraction of RecJ
Growing of S. pombe
Gel electrophoresis on construct 5 and RecJ
PCR on RecQ, RecD2, RecJ, RecA, PprA, Ssb
Genomic extraction of construct 5
Gel electrophoresis on RecQ, RecD2, RecJ, RecA, PprA, Ssb
PCR purification of RecQ, RecD2, RecJ, RecA, PprA, Ssb
Gel electrophoresis on RecJ
10th
Growing of S. pombe
Replated inoculation of construct 1, 2 and 8
2nd transformation of construct 4 into yeast
PCR on purified construct 5 (gDNA)
Gel electrophoresis on construct 5
Transformation of BioBrick vector into E.coli
Preparation of media for Safety Switch testing
11th
PCR on RecJ
Purification of construct 1, 2 and 8 using miniprep
Nanodrop on construct 1, 2 and 8
Restriction of plasmids for construct 1, 2 and 8
Verification of construct 2 and 8
12th
Gel extraction of RecJ
Preparation of chloramphenicol plates
Testing Safety Switch
PCR using PrimeSTAR p0391 (8)
13th
OD measurements of Safety Switch and wildtype
Gel electrophoresis on PCR
PCR of BioBrick a-j04450
Test Safety Switch in YPG
Transformation of pSB1C3 into E .coli
Calculations on Safety Switch test
14th
Gel electrophoresis on PUR samples
Gibson assembly of construct 6, 7 and 8
PCR of Gibson assembly of construct 6, 7 and 8
Gel electrophoresis on construct 6, 7 and 8
Conduction of HPLC on samples from Safety Switch test
PCR purification of BioBrick vector
Restriction of BioBrick vector
Continued test Safety Switch in YPG
18th
Ligation of BioBricks
Transformation of BioBrick and construct 6, 7 and 8 into E.coli
Restriction of BioBrick vector
19th
Ligation of BioBricks
Gel electrophoresis on construct 6, 7 and 8
Re-plating and inoculation of construct 6 and 8
Restriction and ligation of BioBricks
Ligation of BioBrick TPI and CGC-CAS
Transformation of BioBrick TPI and CGC-CAS into E. coli
20th
Purification of construct 6 and 8
Transformation of BioBrick into E. coli
24th
Restriction of construct 6 and 8
Gel electrophoresis on construct 6 and 8
PCR of Safety Switch with RFP and BioBrick Safety Switch
Filtrating S. pombe
25th
Purification of plasmid containing BioBricks (RecA, Ste, Mam, SteMam)
Restriction of BioBricks inserts (RecA, Ste, Mam, SteMam)
Gel electrophoresis on restricted fragments for BioBricks (RecA, Ste, Mam, StêMam)
PCR of Safety Switch RFP
Restriction and ligation of BioBrick TEF and SUC
Gibson assembly of Safety Switch with RFP
26th
Gel electrophoresis of restriction fragments for BioBricks TEF and SUC
Transformation of BioBrick TEFSUC, SUC RFP, TEFSUC RFP into E. coli
Restriction and ligation of BioBricks
Sequencing of parts
27th
3rd transformation of construct 4 into yeast
Gibson assembly of construct 6, 7 and 8 without vector
PCR Gibson assembly of construct 6, 7 and 8 without vector
Transformation of BioBrick (26th) into E. coli
Gibson assembly of TEFSUC RFP and SUC RFP
Purification of BioBrick TEFSUC plasmid
28th
Restriction of construct 4 prior to transformation
2nd transformation of Safety Switch RFP to E. coli
Restriction of BioBrick TEFSUC 1-4
Gel electrophoresis on digested plasmid containing BioBrick TEFSUC 1-4
Restriction of plasmid containing BioBrick TEFSUC1
Gel of plasmid containing BioBrick TEFSUC1 digested with PstI, PstI + EcoRI
30th
Inoculation of construct 4 in CEN.PK2
Inoculation and re-plating of BioBrick TPI, SteMam, RecA, PprA and Ssb
31st
Purification of plasmids containing BioBrick TPI, SteMam, RecA, PprA and Ssb
Restriction of BioBrick TPI, SteMam, RecA, PprA and Ssb
Gel electrophoresis on BioBrick TPI, SteMam, RecA, PprA and Ssb
Re-plating and inoculation of Safety Switch with RFP
Inoculation and plating of construct 4 in CEN.PK2
September
1st
Genomic DNA extraction of construct 4 in CEN.PK2
PCR of construct 4 from CEN.PK2 gDNA
Restriction of TEFSUC insert
Restriction of BioBrick vector
Ligation of BioBrick TEFSUC2
Purification of Safety Switch with RFP using miniprep
Gel electrophoresis on Safety Switch with RFP
Restriction of Safety Switch with RFP
2nd
Gel electrophoresis of construct 4
1st test of construct 4
Transformation of Safety Switch SUC RFP and TEFSUC RFP into yeast CEN.PK2
Restriction of plasmid prior to RFP transformation
Transformation of BioBrick TEFSUC into E. coli
3rd
Concentration of pheromones
Overnight cultivation of CEN.PK2 with m- and p-factor pheromones from S. pombe
Inoculation of construct 4 in E. coli
OD measurements for Safety Switch TPK2
4th
2nd test of Safety Switch TPK2 and WT
Restriction of BioBrick vector and TEFSUC
Ligation of BioBrick TEFSUC with BioBrick vector
7th
Preculture drop test Safety Switch TPK2
Re-plating Safety Switch RFP
8th
Cultivation of cells for spectrometry
PCR of BioBrick TEFSUC and Vector J04450
Preparation pf chloramphenicol plates
Gel electrophoresis on BioBrick TEFSUC and vector J04450
Nanodrop of BioBrick TEFSUC and BioBrick vector
Restriction of BioBrick TEFSUC and BioBrick vector
9th
2nd test of construct 4
Testing BioBricks
Genomic extraction Safety Switch TEF RFP and SUC RFP
Colony PCR Safety Switch TEFRFP and SUC RFP
Gel of Safety Switch RFP and SUC RFP
Colony PCR of BioBrick RFP and SUC RFP
Restriction and ligation of BioBrick TEFSUC
Transformation of BioBrick TEFSUC into E. coli
10th
Restriction of BioBrick and vector
Purification of BioBrick and vector
11th
Concentration of p-factor pheromone
1st droptest on Safety Switch TPK
2nd droptest on Safety Switch TPK
Inoculation of WT, CEN.PK2, Safety Switch TPK2-Col 1, TEFSUC RFP, SUC RFP and RFP
Re-plating and inoculation of E. coli with BioBrick TEFSUC
12th
3rd test of construct 4
Replating of E. coli with BioBrick vector p
14th
Miniprep of E. coli with BioBrick vector from plate
Restriction of product from miniprep (BioBrick vector)
Restriction of BioBrick parts
Gel electrophoresis on cut vector
Ligation of BioBricks in BioBrick vector
Transformation of BioBricks into E. coli and let to grow on plate
Preparation of chloramphenicol plates
16th
Inoculation of E.coli colonies containing BioBricks
17th
Purification of BioBrick using plasmid miniprep kit
Restriction of BioBricks
Gel electrophoresis on BioBricks
18th
Drying of successful BioBricks for submission to iGEM