From 2010.igem.org

Latest revision as of 23:56, 27 October 2010

Parts

• Ribo = Theophylline riboswitch([http://partsregistry.org/Part:BBa_K411001 BBa_K411001])
• RV = Theophylline riboswitch([http://partsregistry.org/Part:BBa_K411001 BBa_K411001]) + pSB1A2
• FRV = Theophylline riboswitch + GFP([http://partsregistry.org/Part:BBa_J04630 BBa_J04630]) + pSB1A2
• PFRV = Theophylline riboswitch + GFP([http://partsregistry.org/Part:BBa_J04630 BBa_J04630]) + pLac([http://partsregistry.org/Part:BBa_R0010 BBa_R0010]) + pSB1A2

2010.08.17

• PCR of primer&Digestion&Ligation

Descr Win length Fail length 1: marker 100bp 2: - - - 3: riboswitch 56 bp none 4: riboswitch 56 bp none 5: - - - 6: riboswitch 56 bp none 7: riboswitch 56 bp none 8: - - - 9: riboswitch 56 bp none 10: riboswitch 56 bp none 11: Positive Control 1100 bp none 12: Negative Control Contamination ~300bp PCR mixfor 3 tubes(25+25+50) total 50 μl temp time template 1μl 94oC 120s forward primer 1μ1 94oC 30s reverse primer 1μ1 55oC 30s dNTP 4μ1 72oC 40s 10x buffer 5μl 72oC 300s ddH2O 39.75μl pfu 0.5μ1

• Plasmid extraction
• Digestion
• PCR purification(centrifuge)
• Nanodrop
• Ligation
• Transform 20:00

2010.08.18

• PCR mix
• Run gel(2%argarose 100v)

	Descr Win length Fail length 1: marker 100bp 2: th-ribo gene + vector 294bp - 3: th-ribo gene + vector 294bp - 4: - - - 5: Positive Control 1100 bp none 6: Negative Control Contamination ~3000bp Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl

(negative contaminated and appeared three different bands)

• 3-in-1 11:30
• Run PCR 12:00
• put Liquid culture&plate at 17:00

2010.08.19

• Because there are some mistake resulting from gel running, we decided to digest again.
  • Digest 10:30
  • Transformation 12:45

• At the same time, we continued plasmid extraction of the result one of yesterday.
  • plasmid extraction 10:30
  • Digest 11:30
  • run gel 13:00
  • Ligation 14:00
  • Transformation 14:30

2010.08.20

• We decide to re-3-in-1 again.
• PCR mix 10:30
• 3 in 1 11:30
• Run PCR 01:00~02:30
• Run gel 15:00(100v 20 mins)

Descr Win length Fail length 1: marker 100bp 2: ribo+vector 294bp 3: ribo+vector 294bp 4: - - - 5: Positive Control 1100 bp none 6: Negative Control Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl Cut gel 16:30 Plasmid extraction 16:30 2010.08.23 the upper experiment are failed, so we restart the experiment. Run gel 11:00 After nanodrop, we found that the concentration is too low(only 3.0), so we re-PCR-of-primer again. PCR of primer 16:00 Transformation(GFP) 17:15 2010.08.24 Run gel 10:30am(5ul) Descr Win length Fail length 1: 2: marker 100bp 3: 4: pcr riboswitch 56bp 5: 6: Positive Control 300bp 7: Negative Control Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl PCR Purify 10:45(45ul) Digest 11:15 Total: 30μl DNA 26μl Digestion buffer 2μl Enzyme 1(X) 1μl Enzyme 2(P) 1μl Digest purify 13:30 Nanodrop(19.8ng/ul) Ligation 15:44 Ligate riboswitch+pSB1A2(1:3) and ligate GFP+pSB1A2(1:2)(we use 2009's GFP+terminater so we have to ligate pSB1A2) Total: 10μl DNA of vector 1μl DNA of insert 3μl buffer 2μl ligase 0.5μl ddH2O 4.5μl Total: 10μl DNA of vector 1μl DNA of insert 2μl buffer 2μl ligase 0.5μl ddH2O 5.5μl 2010.08.25 3-in-1:GFP(1 colony),Riboswitch(2 colony) 17:00pm Run PCR 17:20pm 2010.08.26 Run gel 10:30am Because we thought the result is unsure,we decide to digest to check. Descr Win length Fail length 1: GFP 720bp 2: (1)riboswitch+pSB1A2 294bp 3: (2)riboswitch+pSB1A2 294bp 4: Positive Control 1100bp 5: Negative Control Contamination~1000 bp 6: Marker:100bp Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.25μl Enzyme tag 0.25μl Liquid culture 12:00 the first step when we added MX1 we didn't resuspend,so we readded MX1~MX3.But the DNA may be less. digest [GFP(XP),ribo1(SP,XP>>use 10 ul and 20ul protocal),ribo2(SP,XP>>use 10 ul and use 20ul protocal)] Total: 10μl DNA of vector(sp) 1μl DNA of insert(xp) 3μl buffer 2μl ligase 0.5μl ddH2O 4.5μl run gel(xp) Descr Win length Fail length 1: ribo1 96bp 2: 3: ribo2 96bp 4: 5: Marker:100bp 6: GFP 854bp Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl purify(ribo-1(sp);ribo-2(sp);GFP(xp)) ligate transform liquid culture from the plate(8/24) 2010.08.27 PCR mix 10:30 GFP+terminator+Riboswitch=1157bp 3 in 1 12:15 Run PCR 12:30~13:30 Run gel 13:45 Liquid culture again(use the plate 8/24) 10:30 To check the wrong step of liquid culture Digestion 13:40 Run gel 01:45pm[GFP(XP).Riboswitch-1(XP).Riboswitch-2(XP)] Descr Win length Fail length 1:FRV 1158bp 294bp 2: FRV 1158bp 294bp 3: 100bp marker - - 4: FRV 1158bp 294bp 5: positive control 1100bp 6: Negative Control - Contamination ~300bp Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl 2010.08.28 3-IN-1 (another 3 colony from 08.26 Riboswitch+GFP-Terminator plate) 13:30 PCR (Use new dNTP & Taq buffer & taq) 14:30 Run Gel 16:20 Descr Win length Fail length 1: FRV 1158bp 294bp 2: FRV 1158bp 294bp 3: FRV 1158bp 294bp 4: positive control 1100bp - 5: marker100 bp - - 6: negative control - - Total:(*2) 50μl temp time template 2μl 94oC 60s VF+VR 2μl 94oC 15s dNTP 2μl 55oC 20s Buffer 5μl 72oC 100s ddH2O 39.75μl 72oC 300s Enzyme tag 0.25μl Transformation GFP+vector 17:00 3-IN-1 (2 Colony from the plate transformed on 08/27) 17:20 PCR 18:25~19:40 2010.08.29 Run Gel (with green marker) at 11:00 Ribo Vac1 and Ribo Vac2 from PCR on 8/28 at 18:25~19:40 Descr Win length Fail length 1: - - - 2: - - - 3: RV1 294bp 1100bp 4: marker100 bp - - 5: RV2 294bp - 6: positive control 1100bp - 7: negative control - - Total:(*2) 50μl template 2μl VF+VR dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl ______________________________________________________________________________________________________________ 3-in-1 (3 colonies of GFP+TERMINATOR and 1 of Riboswitch+PSB1A2 vector, both from plates transformed on 8/28) PCR 11:00~12:50 Run Gel 15:30 (result shows no band!! but positive and negative controls are correct) Descr Win length Fail length 1: GFP1 854bp 2: GFP2 854bp 3: GFP3 854bp 4: marker100 bp - - 5: riboswitch+vector 294bp - 6: negative control - - 7: positive contril 1100bp - Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl transform 16:30 (1)the Ribo2 from 8.17 (2)the Ribo (check) from 08/19 (3)GFP+terminator from 08/27 put into incubator 17:30 2010.08.30 3-in-1: GFP,GFP+riboswitch,ribo-1,ribo-2 12:30pm Run PCR 13:20 Run gel(2% argarose 100v) Descr Win length Fail length 1: GFP1 854bp - 2: GFP2 854bp - 3: GFP3 854bp - 4: riboswitch1+GFP 1158bp - 5: riboswitch2+GFP 1158bp - 6: riboswitch3+GFP 1158bp - 7: marker100 bp - - 8: ribo1+vector 294bp - 9: ribo1+vector 294bp - 10: ribo2+vector 294bp - 11: ribo2+vector 294bp - 12: positive control 1100bp - 13: negative control - - Total:(*2) 50μl template 2μl VF+VR dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl Finally we Transform 19F(GFP+terminator), E0040 18:50 2010.08.31 3-in-1 (ribo(xp)+pSB1A2,E0040,J04630)11:00 Nanodrop Ligation Descr Win length Fail length 1: GFP1(J04630) 854bp - 2: GFP2(J04630) 854bp - 3: Riboswitch1+vector 294bp - 4: Riboswitch+2vector 294bp - 5: marker 100 bp - - 6: GFP1(E0040) 720bp - 7: GFP2(E0040) 720bp - 8: ribo1+vector 294bp - 9: ribo1+vector 294bp - 10: ribo2+vector 294bp - 11: ribo2+vector 294bp - 12: positive control 1100bp - 13: negative control - - Total:(*2) 50μl template 2μl VF+VR dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl Descr Win length Fail length 1: GFP+terminator(19f) 854bp - 2: GFP(E0040) 720bp - 3: GFP(E0040) 720bp - 4: marker 100bp - - 5: Positive Control 1100 bp 6: Negative Control 2010.09.01 GFP*3 Plasmid Extraction 10:30 Digestion　12:30 (13:20) Run gel 15:30 Descr Win length Fail length 1: - - - 2: - - - 3: marker 100bp - - 4: - - - 5: (GFP)E0040-2 745bp 6: (GFP)E0040-1 745bp 7: (GFP)J04630 856bp Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.25μl Enzyme tag 0.25μl RG1,RG2,RG3,PR1,PR2,PR3 3-in-1 10:00 Colony PCR 11:00~13:00 Run Gel " Descr Win length Fail length 1: PFRV(1-a) 1158bp 294bp 2: PFRV(1-b) 1158bp 294bp 3: PFRV(2-a) 1158bp 294bp 4: PFRV(2-b) 1158bp 294bp 5: PFRV(3-a) 1158bp 294bp 6: PFRV(3-b) 1158bp 294bp 7: - - - 8: marker 100bp - - 9: PFRV 1366bp - 10: PFRV 1366bp - 11: PFRV 1366bp - 12: PFRV 1366bp f 13: PFRV 1366bp f 14: PFRV 1366bp f 15: positive control 1100bp w 16: negative control - w Total:(*2) 50μl temp time template 2μl 94 60s VF+VR 2μl 94 15s dNTP 2μl 55 20s Buffer 5μl 72 90s ddH2O 39.75μl 72 300s Enzyme tag 0.25μl 2010.09.02 Liquid culture plasmid extraction 10:00 Nanodrop Digestion 12:12 Purification Nanodrop Ligation Transform 17:00 2010.09.03 3-in-1 chose 4 colony PCR 11.20 run gel Descr Win length Fail length 1: FRV1 1158bp 294bp 2: FRV2 1158bp 294bp 3: marker 1kb - - 4: FRV3 1158bp - 5: FRV4 1158bp 294bp 6: positive control 1100bp 7: negative control - Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl (the marker takes the wrong one,1kb marker is correct) ligation failed ligate again(insert+vector=8.5) transform 16:40 2010.09.04 PCR 20:35pm (3 colony from 20100903 plate) run gel(FRV1&2&3&+&-) 100V 35min Descr Win length Fail length 1: FRV1 1158bp 294bp 2: FRV2 1158bp 294bp 3: marker 100bp - - 4: FRV3 1158bp 294bp 5: positive control 1000bp - 6: negative control - - Total:(*2) 50μl template 2μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl discuss 2010.09.05 digest again(because we used XP's protocol for SP) purification at 14:25 nanodrop at 15:00 we digest another tube (ribo2-2 SP) 15:50 ligation transform 17:00 2010.09.06 Colony PCR 11:30 Run gel 14:30(2% argarose 100v 25 mins) Descr Win length Fail length 1: positive control 1100bp - 2: negative control - - 3: 100bp marker - - 4: FRV 1158bp 1700bp 5: FRV 1158bp 1700bp Total:(*2) 50μl template 1μl VF+VR 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl Take out the digest(sp)yesterday 10:30 digest purify nanodrop(13.2ng/nl) ligate 11:40~14:30 transform 17:00 2010.09.07 3-in-1 10:30am Run PCR 10:45am Descr Win length Fail length 1: FRV 1158bp - 2: FRV 1158bp - 3: FRV 1158bp - 4: FRV 1158bp - 5: 100bp marker - - 6: FRV 1158bp - 7: FRV 1158bp 294bp 8: positive control 1100bp - 9: negative control - - Total:(*2) 50μl template 2μl VF+VR dNTP 2μl Buffer 5μl ddH2O 39.75μl Enzyme tag 0.25μl 2010.09.08 plasmid extraction digestion(FRV1(XP)) 11:10 nanodrop 14μl ligation 14:00 Transformation 17:00 2010.09.09 Real PCR Total:(*2) 50μl template 2μl FP+RP 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl pfu 0.25μl Temp Time 94 60s 94 15s 50.8 20s 74 45s 74 300s 2010.09.10 real PCR (temperature test) run gel Total:(*2) 50μl template 2μl FP+RP 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl pfu 0.25μl Temp Time 94 60s 94 15s 50.8 20s 74 45s 74 300s cut gel gel extraction digest(xp) nanodrop ligate transform real pcr for 2 tubes(in suitable temperature) Total:(*2) 50μl template 2μl FP+RP 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl pfu 0.25μl Temp Time 94 60s 94 15s 50.8 20s 74 45s 74 300s 2010.09.11 3in1 two real tubes run gel and cut gel and gel extraction 2010.09.12 run gel(length isn't correct) (real PCR) digestion 12:00 ligation 15:00 transform 19:40 2010.09.13 colony PCR 13:25 (3 in 1) Total:(*2) 50μl template 2μl VR+VF 2μl dNTP 2μl Buffer 5μl ddH2O 39.75μl tag 0.25μl Temp Time 94 60s 94 15s 55 20s 72 30s 30 cycle 72 300s nanodrop ligation 14:00 Colony PCR 15:00 2010.09.22 Real PCR Total: 49μl X1.7 FP 1μl 1.7μl Theophylline Riboswitch Forward Primer RP 1μl 1.7μl Theophylline Riboswitch Reverse Primer dNTP 2μl 3.4μl 10XBuff. 5μl 8.5μl pfu 0.25μl 0.425μl ddH2O 39.75μl 67.575μl PCR Protocol 94 60s 94 15s 52 20s 68 30s 35 cycles 68 300s Digest ribo(xp) overnight Ligate ribo with vector(xp) Transform:23:30 2010.09.23 Ligate ribo with vector again Transform 2010.09.24 3-in-1 (ribo+vector) colony PCR Total: 49μl X1.9 VR+VF 2μl 3.8μl dNTP 2μl 3.8μl 10XBuff. 5μl 9.5μl tag 0.25μl 0.475μl ddH2O 39.75μl 75.525μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s Cut gel(ribo&pSB1A2(xp) digested) 2010.09.26 3-in-1(0925 plate) colony PCR Total: 49μl X2.5 VR+VF 2μl 5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 1.25μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s 2010.09.27 plasmid extraction(RV) Digest RV 13:30 run gel There is GFP on vector, so it's wrong. Digest ribo again overnight 2010.09.28 cut gel(ribo& vector) Ligate ribo with vector Transform According to 0927 gel pic, 30th colony may be correct RV, so we take out second plate and incubate in LB liguid. plasmid extraction 21:30 Digest RV(SP) overnight 2010.09.29 Ligate RV(sp) with GFP(BBa_J04630 already have terminator) Transform 2010.09.30 Transform BBa_J04630 2010.10.01 3-in-1 colony PCR Total: 49μl X1.6 VR+VF 2μl 3.2μl dNTP 2μl 3.2μl 10XBuff. 5μl 8μl tag 0.25μl 0.4μl ddH2O 39.75μl 63.6μl PCR Protocol 94 60s 94 15s 55 20s 72 70s 30 cycles 72 300s 2010.10.02 BBa_J04630 plasmid extraction Digest J04630(XP) cut gel(GFP) Ligate RV(SP) with GFP(XP) Transform 2010.10.03 FRV(RV+GFP) 3-in-1 Total: 49μl X1.9 VR+VF 2μl 3.8μl dNTP 2μl 3.8μl 10XBuff. 5μl 9.5μl tag 0.25μl 0.475μl ddH2O 39.75μl 75.525μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s 2010.10.04 FRV plasmid extraction Digest FRV (XP) cut gel & purify(FRV) Ligate FRV with pLac(from ssrA group) Transform pLac plasmid extraction Digest pLac(SP) overnight 2010.10.05 pLac digest purify(run gel with 3-in-1) PFRV, GFP, RV 3-in-1 Total: 49μl X2.5 VR+VF 2μl 5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 0.625μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s Digest pLac(SP) again overnight 2010.10.06 pLac cut gel Ligate pLac(SP) with FRV(XP) Transform 22:40 Digest FRV(XP) overnight 2010.10.07 FRV(XP) cut gel Ligate FRV with pLac Transform 2010.10.08 PFRV 3-in-1 Total: 49μl X3.8 VR+VF 2μl μl dNTP 2μl 3.8μl 10XBuff. 5μl 9.5μl tag 0.25μl 0.475mu;l ddH2O 39.75μl 75.525μl PCR Protocol 94 60s 94 15s 55 20s 72 100s 30 cycles 72 300s Ligate FRV with pLac Transform 22:15 2010.10.09 PFRV 3-in-1 Total: 49μl X3.8 VR+VF 2μl 7.6μl dNTP 2μl 7.6μl 10XBuff. 5μl 17μl tag 0.25μl 0.95μl ddH2O 39.75μl 151.06μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s colony PCR again Total: 49μl X3.8 VR+VF 2μl 7.6μl dNTP 2μl 7.6μl 10XBuff. 5μl 17μl tag 0.25μl 0.95μl ddH2O 39.75μl 151.06μl PCR Protocol 94 60s 94 15s 55 20s 72 180s 30 cycles 72 300s Ligate FRV with pLac again Transform 2010.10.10 PFRV 3-in-1 Total: 49μl X3.2 VR+VF 2μl 6.4μl dNTP 2μl 6.4μl 10XBuff. 5μl 15μl tag 0.25μl 0.8μl ddH2O 39.75μl 127.2μl PCR Protocol 94 60s 94 15s 55 20s 72 180s 30 cycles 72 300s length is correct!!! F colony liquid culture FRV liquid culture Digest FRV(XP) again 2010.10.11 FRV&PFRV plasmid extraction FRV cut gel Digest PFRV(XP)&FRV&pSB1C3 Ligate PFRV with C3/ FRV with C3/ ribo with C3 Transform 2010.10.12 Ligate PFRV with C3/FRV with C3/ribo with C3 again Transform 17:25 2010.10.13 PFRC&FRC&RC 3-in-1 Total: 49μl X1.7 VR+VF 2μl 3.4μl dNTP 2μl 3.4μl 10XBuff. 5μl 8μl tag 0.25μl 0.425μl ddH2O 39.75μl 67.575μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s Ligate PFRV,FRV,ribo with C3 Transform 00:50 Digest PFRV(XP)&RV(XP) again overnight 2010.10.14 PFRV&RV run gel Ligate RV,FRV,PFRV with C3 again Transform 2010.10.15 FRC,RC 3-in-1 Total: 49μl X2.5 VR+VF 2μl 5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 0.625μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s 3-in-1 again Total: 49μl X2.5 VR+VF 2μl 5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 0.625μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s FRC and PFRC are correct, but RC is still incorrect PFRC&FRC liquid culture Ligate ribo with C3 again Transform 2010.10.16 Theophylline solution Take 1.8012g Theophylline into 15ml DMSO;concentration is 0.67M put 15 μl into LB liquid, concentration is 2.5mM; put 30 μl into LB liquid, concentration is 5mM Ligate ribo(xp) with C3 again Transform 2010.10.17 transform second plate of PFRC into agar plate positive:C3 30μl+theophylline 0.0027g negative:C3 PFRC&FRC plasmid extraction 2010.10.18 PFRC PCR test Total: 49μl X1.3 VR+VF 2μl 2.6μl dNTP 2μl 2.6μl 10XBuff. 5μl 6.5μl tag 0.25μl 0.325μl ddH2O 39.75μl 51.675μl PCR Protocol 94 60s 94 15s 55 20s 72 90s 30 cycles 72 300s Theophylline in DMSO add 1 ml DMSO + Theophylline into 4 ml LB Ligate ribo with C3 again Transform Digest ribo(XP)& C3(XP) ribo & C3 cut gel 2010.10.19 Assay Test O.D/2 O.D add to liquid PFRC 2.503 5.006 166.5 LB 0 0 Because Theophylline entry into E. coli spend 1 hours at least, O.D= 0.0325 Assay:add 80 μl PFRC liquid into LB Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl) A 0 mM 0 80.4 4 B 0.5 mM 2.985 μl 80.4 4 C 1 mM 5.97 μl 80.4 4 D 2 mM 11.94 μl 80.4 4 E 4 mM 23.88 μl 80.4 4 F 5 mM 29.85 μpl 80,4 4 10/18 RC 3-in-1 Total: 49μl X2.1 VR+VF 2μl 4.2μl dNTP 2μl 4.2μl 10XBuff. 5μl 10.5μl tag 0.25μl 0.525μl ddH2O 39.75μl 83.475μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s PFRC & C liquid culture Ligate ribo with C3 again Transform 2010.10.20 Assay Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl) O 0 mM 0 130 4 A 0.1 mM 0.597 μl 130 4 B 0.05 mM 1.4925 μl 130 4 C 0.5 mM 2.985 μl 130 4 D 1 mM 5.97 μl 130 4 E 2 mM 11.94 μl 130 4 F 4 mM 23.88 μpl 130 4 G 8 mM 47.76 μl 130 4 culture for 2 hours Transform 2010.10.21 Assay Theophylline concentration Theophylline(0.67M) PFRC liquid(μl) Cm50(μl) O 0 μM 0 85.7 4 A 50 μM 0.2985 μl 85.7 4 B 100 μM 0.597 μl 85.7 4 C 200 μM 1.194 μl 85.7 4 D 500 μM 2.985 μl 85.7 4 E 1000 μM 5.97 μl 85.7 4 F 2000 μM 11.94 μl 85.7 4 G 4000 μM 23.88 μl 85.7 4 H 8000 μM 47.76 μl 85.7 4 I 10000 μM 59.7 μl 85.7 4 J 20000 μM 119.4 μl 85.7 4 K 40000 μM 238.8 μl 85.7 4 10/20 RC 3-in-1 Total: 49μl X2.5 VR+VF 2μl 5μl dNTP 2μl 5μl 10XBuff. 5μl 12.5μl tag 0.25μl 1.25μl ddH2O 39.75μl 99.375μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s Digest ribo again 2010.10.22 Digest C3 from PFRC plasmid Ligate ribo with C3 Transform Preparing 0.1 M Theophylline solution in DMSO. Assay Theophylline concentration Theophylline(0.1M) PFRC liquid(μl) Cm50(μl) O 0 μM 0 85.7 4 A 10 μM 0.4 μl 117.7 4 B 50 μM 2 μl 117.7 4 C 100 μM 4 μl 117.7 4 D 200 μM 8 μl 117.7 4 E 500 μM 20 μl 117.7 4 F 1000 μM 40 μl 117.7 4 G 2000 μM 80 μl 117.7 4 H 4000 μM 160 μl 117.7 4 I 8000 μM 320 μl 117.7 4 J 10000 μM 400 μl 117.7 4 K 20000 μM 800 μl 117.7 4 Modified experiment design Theophylline concentration Theophylline(0.1M) PFRC liquid(μl) FRC liquid(μl) Cm50(μl) O 0 μM 0 261 0 4 A 10 μM 0.4 μl 261 0 4 B 50 μM 2 μl 261 0 4 C 100 μM 4 μl 261 0 4 D 200 μM 8 μl 261 0 4 E 500 μM 20 μl 261 0 4 F 1000 μM 40 μpl 261 0 4 G 2000 μM 80 μl 261 0 4 H 4000 μM 160 μl 261 0 4 I 8000 μM 320 μl 261 0 4 J 10000 μM 400 μl 261 0 4 K 20000 μM 800 μl 261 0 4 NT 100 μM 4 μl 0 261 4 N 0 μM 0 μl 0 261 4 2010.10.24 ribo 3-in-1 Total: 49μl X0.9 VR+VF 2μl 1.8μl dNTP 2μl 1.8μl 10XBuff. 5μl 4.5μl tag 0.25μl 0.225μl ddH2O 39.75μl 35.775μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s ribo liquid culture colony PCR Total: 49μl X1.6 VR+VF 2μl 3.2μl dNTP 2μl 3.2μl 10XBuff. 5μl 8μl tag 0.25μl 0.8μl ddH2O 39.75μl 63μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s Assay Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl) FRC liquid(μl) Cm50(μl) O 0 μM 0 325 325 0 4 A 10 μM 0.4 μl 325 325 0 4 B 50 μM 2 μl 327 325 0 4 C 100 μM 4 μl 329 325 0 4 D 200 μM 8 μl 333 325 0 4 E 500 μM 20 μl 345 325 0 4 F 1000 μM 40 μpl 365 325 0 4 G 2000 μM 80 μl 405 325 0 4 H 4000 μM 160 μl 485 325 0 4 I 8000 μM 320 μl 645 325 0 4 J 10000 μM 400 μl 725 325 0 4 K 20000 μM 800 μl 1125 325 0 4 NT 100 μM 4 μl 504 0 500 4 N 0 μM 0 μl 500 0 500 4 Digest RV(XP)& FRC(XP) Ligate ribo with C3 transform PFRC&FRC liquid culture 2010.10.25 RC 3-in-1 Total: 49μl X2.4 VR+VF 2μl 4.8μl dNTP 2μl 4.8μl 10XBuff. 5μl 12μl tag 0.25μl 0.6μl ddH2O 39.75μl 95.4μl PCR Protocol 94 60s 94 15s 55 20s 72 30s 30 cycles 72 300s FRC&PFRC&RC liquid culture Assay Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl) FRC liquid(μl) Cm50(μl) O 0 μM 0 151 147 0 4 A 50 μM 2 μl 153 147 0 4 B 500 μM 20 μl 171 147 0 4 C 1000 μM 40 μl 191 147 0 4 D 4000 μM 160 μl 311 147 0 4 E 10000 μM 400 μl 551 147 0 4 NT 100 μM 4 μl 158 0 150 4 N 0 μM 0 μl 154 0 150 4 2010.10.26 Assay Theophylline concentration Theophylline(0.1M) Remove LB(μl) PFRC liquid(μl) FRC liquid(μl) Cm50(μl) O 0 μM 0 151 147 0 4 A 50 μM 2 μl 153 147 0 4 B 500 μM 20 μl 171 147 0 4 C 1000 μM 40 μl 191 147 0 4 D 4000 μM 160 μl 311 147 0 4 E 10000 μM 400 μl 551 147 0 4 NT 100 μM 4 μl 158 0 150 4 N 0 μM 0 μl 154 0 150 4 NYMU-Taipei iGEM 2010 Team