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RecA: July 10 - September 28
From 2011.igem.org
7/9 Saturday • added DPN1 to RecA1 mutegensis • incubated 4-6 hours • PCR purfified • transfromed RecA1: o A3: RecA1 III from 7/7 T.C.: 5.0 o A5 : RecA1 V from 7/7 T.C.:5.2 o B3: : RecA1 III from 7/8 T.C: 5.0 o B5: : RecA1 V from 7/8 T.C. 5.0 • Transfomred test: T.C.: 2.2 • plated transformations and made freezer stock of RecA1III 7/10 Sunday • got sequencing results back of RecA1 III and V. III is good even when blasting with NCBI, • V is definetly reversed • test did not grow • ran gel of its PCR purification think had to much lyse because came out smeared just below 1000bp • began digest of PCR purfication of part 2 from 7/8 miniprep of part 1 from ⅞ • litgate • transformed • plate 7/11Monday • centrifuge broke • nothing grew of test plates 7/12 Tuesday • centrifuge fixed. Anisha miniprep RecaA1 • grew Part 1 and Part 2 from registry 7/13 Wednesday • part cultures did not grow because I forgot to transform • transformed from registry o part 1 T.C.: 2.8 o Part 2 T.C.: 2.6 o cultured • RecA1 mute results back--> mutation didn’t work • New mutegeneiss PCR with extention time 2min 15 seconds to go overnight 7/14Thursday • added DPN1 to RecA1 mute PCR, left to incubate • transformed o T.C.: 5.0 • plated • mini prep part 1 and part 2 • PCR insert Part 2 • digested • ligated • transformed: T.C.: 2.2 • Byron started another PCR of RecA1 mute to run over night 7/15Friday • colonies grew of test and RecA1 but very small so will let grow one more night and PCR colony the next day • Byron o added DPN1 and incubated for 6 hours o heat killed at 80 degree fro 20 min (this should not have happened and probably why mutegensisi didn’t work) o PCR purified product o transformed T.C.: 2.0 o plated 7/16 Saturday • colony PCR test: 2 from each plate 1A, 1B, 2A, 2B, 3A, 3B nothing grew on plate 4 • made culture of RecA1 mute 3 from one plate : 1A, 1B, 1C. Other plate only had one colony • forgot to add oil before PCRing, started another PCR whil cultureing all sample 7/18 • mini-prepped RecA1 Ecor1 Pst1 mutegensis cultures A and B • sent the test to sequencing. Terminator came out but the driver didn’t show up. Remu did not e • going to assemble test again using B0015 mini-prep from 7/14 going to PCR driver from registry • also going to try to assemble with Terminator as the insert and the driver as the vector. Terminator insert digest with E&S enzymes and Vector digest with E&X 7/19 • test using mini-prep from 7/14 of terminator and registry assembled with terminator as vector and driver as insert • PCR driver • Digest • Ligate • Transformed o Time constant 2.0 • Plated • Also assembled mini-prep terminator and mini-prep driver 7/14 but with terminator as insert and driver as insert • PCR terminator • Digest using buffer 4 • Ligate • Transformed o T.C. 2.0 • Plated A&B with assemble of terminator as insert and driver as vector • Plated C&D with assemble of terminator as driver and driver as vector RecA1 Mutagenesis • Multisite mutagenesis was conducted for EcoR1 and Pst1 • Protocol followed was the desired mutagenesis from open wetware • Also ran mutagenesis adjusting the original multisite muteagenesis protocol o MgSO4: 0, 0.5, 1 ul o DMSO: 0, 0.5, 1 o Without DMSO with primer • Let PCR run overnight Mutagenesis—multisite protocol varying MgSO4 and addition of Reverse Primer: • With reverse primer o 1: 0ul MgSO4 o 2: 0.5ul MgSO4 o 3: 1ul MgSO4 • Without reverse Primer o 4: 0ul MgSO4 o 5: 0.5ul MgSO4 o 6: 1ul MgSO4 • Due to supplies was only able to do one mutagenesis, choose to do 4 7/20 • PCR colony tests • Cultured test RecA • Incubated 4 hours • PCR purified RecA1 mutagenesis • Transformed o T.C. 4.4 • Plated • Reviewed sequencing results from 7/18, came back unsuccessful 7/21 • Mini-prep RecA1 send to sequencings • Culture RecA1 mute1 • Mini-prep terminator and driver and RecA1 III • Also made freezer stock • PCR insert of terminator with extension time 45 seconds • Plated two colonies (I, II) from transformation on 7/19 (PCR without reverse primer) • Made culture 7/22 • No centrifuge tubes so unable to send to sequencing • PCR purified terminator • Digested terminator as insert and driver as vector • Ligated • Transformed o T.C. 1.8 • Mini-prepped culture (I and II) from 7/21 and sent to sequencing • Began other mutagenesis’s by beginning of PCR o 1, 2, 3, 5, and 6 7/23 • Incubated Mutagenesis’s for 4 hours • PCR purified • Transformed o T.C. 1: 4.4 2: 4.8 3: 4.8 5: 4.8 6: 4.0 7/24 • Colonies were not very big waiting until Monday • Began another RecA mutagenesis starting with PCR o Primers Pst, Arg243, Lys 286 • Minipreped mutagenesis 4 and sent to sequencing 7/25 • Cultured colonies from test 1A, 1B, 2A, 2B, 3A, 3B, 5A, 5B, 6A, ^B and test • Reviewed sequencing: unsuccessful • Incubated second RecA mutagenesis • PCR purified • Transformed 7/26 • Minipreped colonies however was late to sequencing • Began a third mutageneis with different amounts of DMSO and PCR annealing temperature 7/27 • Cultured test again • Sequencing results had a lot of Ns • Started new test assembly o 1: Vector = Driver Enzymes E&X, Insert=Terminator, Enzymes E&S o PCRed, ligated, transformed (T.C. 2.4) palted • Incubated RecA • PCR purfified • Transformed (T.C: 5.2) • plated 7/28 • Pst primer and EcoR1 primers were re-ordered because discovered they were incorrect • No colonies grew on test plate • Grew terminator and driver from freezer stock 7/29 • Cultured RecA from plates • Mini-prepred terminator and driver and continued assembly (T.C. 3.2) • Mini-prep RecA mutagenesis and sent to sequencing 7/30 • cultured test assembly • reviewed RecA results: unsuccessful 7/31 • started new RecA mutagenesis o varying concentrations and PCR temperatures • mini-prep test and sent to sequencing 8/1 • continued RecA mutagenesis • reviewed test sequencing 8/2 • reviewed test and RecA mini-preps using a nanodrop because suspicion of bad sequencing read • several did not turn out well so re-mini-prepped and sent to sequencing 8/3 • checked sequencing still unsuccessful 8/4 • discovered mini-prep test kit was contaminated • retrieved new kit and began mutagenesis 8/8 • Began test assembly • Using terminator and driver from 7/18 (had be verified through sequencing that it was terminator and driver) • Digest o Driver as vector enzymes E&X o Terminator as insert E&S • Ligated • Transformed ligation o T.C.: 2.2 • Plated 8/9 • Cultured parts for new assembly • Colony PCR product from 8/8 8/12 • Cultured test, terminator and driver 8/13 • Miniprepped from 8/12 9/8 - Using CBAR for mutagenesis - Designed oligos -> ordered - PCR’d boh B0015 and I763007 9/9 - Oligo arrived -> ran PCR - Rec A Lys Mutation o Pst Forward, EcoR1 Reverse o Pst R, EcoR1 F - RecA Pst Mutagenesis o Arg F, Lys R o Arg R, Lys F - PCR Purified – B0015 and I763007 - Digested o Term Vector = S, P Insert = E, S o Driver Vector = E, X Insert = X, P - Digest Purified Vectors - Ligated o Test 1 = Term (V) + Driver (I) o Test 2 = Term (I) + Driver (V) - Transformed o Test 1 = 3.0 o Test 2 = 3.0 - Plated o A plates 9/12 - PCR Purified - Nanodropped o 14.6 ng/µL o 19.8 o 41.5 o 9.7 - Fentamole Calculations were done - Waited to verify calculation with supervisor - Diluted PCRs to correct concentration - Combined the following together w/ CBAR mixture: o Pst F + EcoR1 R with Pst R + EcoR1 F o Arg F + Lys R with Arg R + Lys F - Incubated at 50 degrees C for 1 hour. - PCR purified - Transformed .5 µL on K resistance - Plated - PCR Colony – Test 1 and Test 2 - Ran on gel o Looked good - Cultured - Transformed 2nd half of circuit o T.C. =5.2 - Cultured part 9/13 - Miniprepped test 1, 2, and the second half of the circuit (B) - Sent to sequencing 9/14 - Colony stab and cultured - Read sequencing results – Test 1, 2, B o Test 1 = not good o Test 2 = GOOD! :D o Test B = GOOD! :D o PCR’d all parts that worked 9/15 - CBAR Epic fail - Ran PCR product on gel o Looked good - PCR Purified - Digested o Part 1 (Test 2) Vector = S,D Insert = E, S o Part 2 (Test B) Vector = E, X Insert = X, P - Ligated o Test 1 = Part 1 (V) + Part 2 (I) o Test 2 = Part 2 (V) + Part 1 (I) - Transformed o Test 1 = 2.8 o Test 2 = 2.6 - Plated o A Resistence 9/16 - Test 1 nor Test 2 grew – started over 9/19 - Mutagenesis o PE + Arg -> fail o PE + Lys -> worked o LE + Arg -> fail o LE + Pst1 -> fail
