Difference between revisions of "Team:Aachen/Notebook/Documentation/Methanol Single Expression"
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! construct description !! sequencing confirmed plasmid !! confirmed cryo | ! construct description !! sequencing confirmed plasmid !! confirmed cryo | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh in pSB1A30 || #BZBQ# || #IGEM# |
|- | |- | ||
− | | RBS. | + | | RBS.hps in pSB1A30|| #4PAB# || #4P9H# |
|- | |- | ||
− | | RBS. | + | | RBS.phi in pSB1A30 || #PEA6# || #TFE1# |
|- | |- | ||
− | | RBS. | + | | RBS.xpk in pSB1A30|| #1NFT# || #ONK4# |
|} | |} | ||
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'''restrictions:''' | '''restrictions:''' | ||
# B0015 in pSB1C3 #TK6C# with XbaI and PstI | # B0015 in pSB1C3 #TK6C# with XbaI and PstI | ||
− | # | + | # hps in pSB1C3 #H4M9# with EcoRI and SpeI |
− | # | + | # phi in pSB1C3 #ANNA# with EcoRI and SpeI |
''calculation:'' | ''calculation:'' | ||
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! Volumes for (1.) B0015 [µl] !! Volumes for (2.) | + | ! components !! Volumes for (1.) B0015 [µl] !! Volumes for (2.) hps [µl] !! Volumes for (3.) phi [µl] |
|- | |- | ||
| ddH{{sub|2}}O || 14.7 || 15.2 || 15.5 | | ddH{{sub|2}}O || 14.7 || 15.2 || 15.5 | ||
Line 43: | Line 43: | ||
* restriction products stored in Falcon #TUSS# | * restriction products stored in Falcon #TUSS# | ||
'''ligation:''' | '''ligation:''' | ||
− | * '''(A)''' (1.) B0015 digest and (2.) | + | * '''(A)''' (1.) B0015 digest and (2.) hps digest into pSB1K30 digest (#SZKD#) |
− | * '''(B)''' (1.) B0015 digest and (3.) | + | * '''(B)''' (1.) B0015 digest and (3.) phi digest into pSB1K30 digest (#SZKD#) |
{|class="wikitable" | {|class="wikitable" | ||
! components !! Volumes for '''(A)''' [µl] !! Volumes for '''(B)''' [µl] | ! components !! Volumes for '''(A)''' [µl] !! Volumes for '''(B)''' [µl] | ||
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'''restriction''' | '''restriction''' | ||
− | * | + | *xpk in pSB1C3 (#KFEY#) with EcoRI and SpeI (X) |
− | * | + | *mdh in pSB1C3 (#9PBD#) with EcoRI and SpeI (M) |
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! Volumes for Xpk (X) [µl] !! Volumes for | + | ! components !! Volumes for Xpk (X) [µl] !! Volumes for mdh (M) [µl] |
|- | |- | ||
| ddH{{sub|2}}O || 16.5 || 15.4 | | ddH{{sub|2}}O || 16.5 || 15.4 | ||
Line 86: | Line 86: | ||
|} | |} | ||
− | * | + | *xpk restictions: tube X |
− | * | + | *mdh restrictions: tube M |
'''ligation''' | '''ligation''' | ||
− | * '''(A)''' B0015 digest from #OHOZ# and (2) | + | * '''(A)''' B0015 digest from #OHOZ# and (2) hps digest from #TUSS# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(B)''' B0015 digest from #OHOZ# and (3) | + | * '''(B)''' B0015 digest from #OHOZ# and (3) phi digest from #TUSS# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(C)''' B0015 digest from #OHOZ# and (X) | + | * '''(C)''' B0015 digest from #OHOZ# and (X) xpk digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD# |
− | * '''(D)''' B0015 digest from #OHOZ# and (M) | + | * '''(D)''' B0015 digest from #OHOZ# and (M) mdh digest from #OHOZ# into (K30) pSB1K30 digest from #SZKD# |
{|class="wikitable" | {|class="wikitable" | ||
Line 104: | Line 104: | ||
| upstream Part Digest || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) | | upstream Part Digest || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) || 4 (B0015 digest B0015 from #OHOZ#) | ||
|- | |- | ||
− | | downstream Part Digest || 4 ( | + | | downstream Part Digest || 4 (hps digest 2 from #TUSS#) || 4 ((phi digest 3 from #TUSS#) || 4 (xpk digest X from #OHOZ#) || 4 (mdh digest M from #OHOZ#) |
|- | |- | ||
− | | pSB1K30 digest ( | + | | pSB1K30 digest (plasmid from #SZKD#) || 2 || 2 || 2 || 2 |
|- | |- | ||
| T4 DNA Ligase || 1 || 1 || 1 || 1 | | T4 DNA Ligase || 1 || 1 || 1 || 1 | ||
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==15-06-18== | ==15-06-18== | ||
− | * make | + | * make overnight cultures and masterplates |
** 12 clones of each construct were plated on master plates. 6 overnights were made. | ** 12 clones of each construct were plated on master plates. 6 overnights were made. | ||
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! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | | + | | mdh.B0015 in pSB1K30 clones 1-12 || 37-48 || 1682 || 2 || 1'50" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | hps.B0015 in pSB1K30 clones 1-12 || 13-24 || 1160 || 1 || 1'13" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | phi.B0015 in pSB1K30 clones 1-12 || 1-12 || 1079 || 1 || 1'13" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|- | |- | ||
− | | | + | | xpk.B0015 in pSB1K30 clones 1-12 || 73-84 || 3002 || 3 || 3'05" || Negative for all clones. Observed bands at 1227 bp (RFP) |
|} | |} | ||
* We picked only "red" clones, because we forgot to induce with IPTG. | * We picked only "red" clones, because we forgot to induce with IPTG. | ||
− | * | + | * with green light the red flourescence could be observed very well |
* we picked the few white clones and put them on a masterplate LB+K | * we picked the few white clones and put them on a masterplate LB+K | ||
* next step will be a new colony PCR on monday. | * next step will be a new colony PCR on monday. | ||
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! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | | + | | mdh.B0015 in pSB1K30 clones 1-3 || 6-8 || 1682 || 2 || 1'50" || bands are too short |
|- | |- | ||
− | | | + | | hps.B0015 in pSB1K30 clones 1-3 || 1-3 || 1160 || 1 || 1'13" || bands are too short |
|- | |- | ||
− | | | + | | phi.B0015 in pSB1K30 clones 1-2 || 4-5 || 1079 || 1 || 1'13" || bands are too short |
|- | |- | ||
− | | | + | | xpk.B0015 in pSB1K30 clones 1-3 || 9-11 || 3002 || 3 || 4'00" || bands are too short |
|} | |} | ||
* restriction/ligation didn't work out as we expected. | * restriction/ligation didn't work out as we expected. | ||
− | * problem: pSB1K30 digest | + | * problem: pSB1K30 digest never works properly |
** next step: plate cryo #8T49# to get more plasmid of pSB1K30 | ** next step: plate cryo #8T49# to get more plasmid of pSB1K30 | ||
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|} | |} | ||
* ligate each RBS.CDS digest with the B0015 out of pSB1C3 into pSB1A30 digest | * ligate each RBS.CDS digest with the B0015 out of pSB1C3 into pSB1A30 digest | ||
+ | |||
{|class="wikitable" | {|class="wikitable" | ||
− | ! components !! (A) RBS. | + | ! components !! (A) RBS.mdh.B0015 in pSB1A30 volumes [µl] !! (B) RBS.hps.B0015 in pSB1A30 volumes [µl] !! (C) RBS.phi.B0015 in pSB1A30 volumes [µl] !! (D) RBS.xpk.B0015 in pSB1A30 volumes [µl] |
|- | |- | ||
| H{{sub|2}}O || 7 || 7 || 7 || 7 | | H{{sub|2}}O || 7 || 7 || 7 || 7 | ||
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|} | |} | ||
ligation products stored in falcon #CAJO# | ligation products stored in falcon #CAJO# | ||
− | * heatshock transformation into BL21. Don't forget | + | * heatshock transformation into BL21. Don't forget induction with IPTG for red/white screening! |
==15-06-24== | ==15-06-24== | ||
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! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 1 || 1 || 1682 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 1-3 || 2, 3, 4 || 1160 || 2 || 0'25'' || no bonds |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 1-6 || 5, 6, 7, 8, 9, 10 || 1079 || 2 || 0'25'' || very bad bands |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1-3 || 11, 12, 13 || 3002 || 5 || 0'45'' || very bad bands |
|} | |} | ||
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! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 || 1-4 || 1682 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 3 || 5 || 1160 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 || 6-16|| 1079 || 2 || 0'25'' || no bands |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1, 7 || 17, 18 || 3002 || 5 || 0'45'' || no bands |
|} | |} | ||
− | *control with red clones: | + | *control with red clones: mdh (tube MR), hps (tube HR), phi (tube PR), xpk (XR) |
'''We have to think about a new strategy, as the Biobrick assembly seems not to work''' | '''We have to think about a new strategy, as the Biobrick assembly seems not to work''' | ||
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In a next step we will swap the RBS.CDS.B0015 constructs into pSB1K30 backbones. | In a next step we will swap the RBS.CDS.B0015 constructs into pSB1K30 backbones. | ||
− | Monday and Wednesday are the days with the most workload, while tuesday and thursday give space to plan further steps | + | Monday and Wednesday are the days with the most workload, while tuesday and thursday give space to plan further steps |
* cut #FW6L# with EcoRI and XbaI; reaction volume: 50 µl; water: 36.3 µl; DNA: 6.7 µl | * cut #FW6L# with EcoRI and XbaI; reaction volume: 50 µl; water: 36.3 µl; DNA: 6.7 µl | ||
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* digest RBS.CDS constructs with EcoRI and PstI | * digest RBS.CDS constructs with EcoRI and PstI | ||
− | ** RBS. | + | ** RBS.mdh from #YSVQ# |
− | ** RBS. | + | ** RBS.hps from #H4M9# |
− | ** RBS. | + | ** RBS.phi from #ANNA# |
− | ** RBS. | + | ** RBS.mdh from #KFEY# |
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* control digests on gel | * control digests on gel | ||
*results | *results | ||
− | *** #TUSS#: | + | *** #TUSS#:mdh (expected: 1220 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:hps (expected: 698 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:phi (expected: 617 bp / 2037 bp): bands at expected length |
− | *** #TUSS#: | + | *** #TUSS#:xpk (expected: 2540 bp / 2037 bp): bands at expected length |
*** #TUSS#:K30 (expected: 902 bp / 2222 bp): no bands at all | *** #TUSS#:K30 (expected: 902 bp / 2222 bp): no bands at all | ||
* ligation of fragments | * ligation of fragments | ||
** Ligation of #OHOZ#:1 with ... | ** Ligation of #OHOZ#:1 with ... | ||
− | *** #TUSS#: | + | *** ...#TUSS#:mdh (ML) |
− | *** #TUSS#: | + | *** ...#TUSS#:hps (HL) |
− | *** #TUSS#: | + | *** ...#TUSS#:phi (PL) |
− | *** #TUSS#: | + | *** ...#TUSS#:xpk (XL) |
− | Pipeting volumes for | + | Pipeting volumes for ligation |
{|class="wikitable" | {|class="wikitable" | ||
! component !! volume [µl] | ! component !! volume [µl] | ||
Line 336: | Line 337: | ||
| T4 Ligase || 1 | | T4 Ligase || 1 | ||
|} | |} | ||
− | * ligation products stored in | + | * ligation products stored in falcon #LIGJ# |
− | * | + | * transformation into BL21 Gold (DE3) |
==15-07-01== | ==15-07-01== | ||
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! gene / clones !! tube number / row in gel !! expected length !! machine !! elongation time !! result | ! gene / clones !! tube number / row in gel !! expected length !! machine !! elongation time !! result | ||
|- | |- | ||
− | | RBS. | + | | RBS.mdh.B0015 in pSB1A30 clones 6, 8, 10, 11 || 1-4 || 1682 || 2 || 0'25'' || no positive clones |
|- | |- | ||
− | | RBS. | + | | RBS.hps.B0015 in pSB1A30 clones 3 || 5 || 1160 || 2 || 0'25'' || no positive clones |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 clones 2, 4, 6, 7, 8, 13, 17-21 || 6-16|| 1079 || 2 || 0'25'' || clones 13, 19, 20 and 21 positive |
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 clones 1, 7 || 17, 18 || 3002 || 5 || 0'45'' || clone 7 positive |
|} | |} | ||
====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ||
− | * make masterplates and | + | * make masterplates and overnight cultures |
====RBS.CDS in pSB1A30 (ML, HL, PL, XL)==== | ====RBS.CDS in pSB1A30 (ML, HL, PL, XL)==== | ||
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====RBS.CDS.B0015 in pSB1A30==== | ====RBS.CDS.B0015 in pSB1A30==== | ||
* purify plasmid of: | * purify plasmid of: | ||
− | ** | + | ** xpk clone 7 |
− | ** | + | ** phi clones 13, 19, 20, 21 |
====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ====RBS.CDS.B0015 in pSB1A3 (E, F, G, H)==== | ||
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! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ||
|- | |- | ||
− | | E || RBS. | + | | E || RBS.mdh.B0015 in pSB1A3 || 1-8 || 1631 || 0'25'' || nothing |
|- | |- | ||
− | | F || RBS. | + | | F || RBS.hps.B0015 in pSB1A3 || 9-16 || 1109 || 0'25'' || nothing |
|- | |- | ||
− | | G || RBS. | + | | G || RBS.phi.B0015 in pSB1A3 || 17-24 || 1028 || 0'25'' || nothing |
|- | |- | ||
− | | H || RBS. | + | | H || RBS.xpk.B0015 in pSB1A3 || 25-32 || 2951 || 0'45'' || clone 26 positive |
|} | |} | ||
* purify plasmid and make cryos of positive clones | * purify plasmid and make cryos of positive clones | ||
Line 382: | Line 383: | ||
− | * because | + | * because almost nothing worked: new transformation of ligation products #CAJO#:E/F/G/H |
** 50 µl competent DH5alpha | ** 50 µl competent DH5alpha | ||
** 2 µl ligation product | ** 2 µl ligation product | ||
Line 392: | Line 393: | ||
! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ! construct ID !! construct description !! clone numbers !! expected length [bp] !! elongation time !! result | ||
|- | |- | ||
− | | ML || RBS. | + | | ML || RBS.mdh in pSB1A30 || 33-40 || 1553 || 0'25'' || Clone: 34, 35, 37, 39 |
|- | |- | ||
− | | HL || RBS. | + | | HL || RBS.hps in pSB1A30 || 41-48 || 1031 || 0'25'' || Clone: 41 - 48 |
|- | |- | ||
− | | PL || RBS. | + | | PL || RBS.phi in pSB1A30 || 49-56 || 950 || 0'25'' || Clone: 49 - 56 |
|- | |- | ||
− | | XL || RBS. | + | | XL || RBS.xpk in pSB1A30 || 57-64 || 2873 || 0'45'' || clones 57 and 62 positive |
|} | |} | ||
* purify plasmid and make cryos of positive clones | * purify plasmid and make cryos of positive clones | ||
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! Construct !! Clone !! Plasmid ID !! Cryo ID !! Plasmid konzentration !! Sequencing result | ! Construct !! Clone !! Plasmid ID !! Cryo ID !! Plasmid konzentration !! Sequencing result | ||
|- | |- | ||
− | | H: RBS. | + | | H: RBS.xpk.B0015 in pSB1A3 || 26 || #8ZRY#|| #84BM# || 234.7 || VALID |
|- | |- | ||
− | | ML: RBS. | + | | ML: RBS.mdh in pSB1A30 || 34 || #1RZF# || #8AZD#|| 136.5 || discard |
|- | |- | ||
| || 35 || #PZRX# ||#MB3A# || 160.3 || VALID | | || 35 || #PZRX# ||#MB3A# || 160.3 || VALID | ||
Line 416: | Line 417: | ||
| || 39 || #6WTB# || #HEXE# || 133.8 || discard | | || 39 || #6WTB# || #HEXE# || 133.8 || discard | ||
|- | |- | ||
− | | HL: RBS. | + | | HL: RBS.hps in pSB1A30 || 41 || #YSAE# || #MKCP# || 165.4 || VALID |
|- | |- | ||
| || 42 || #T8KB# || #DR63# || 133.4 || discard | | || 42 || #T8KB# || #DR63# || 133.4 || discard | ||
Line 424: | Line 425: | ||
| || 44 || #4PAB# || #4P9H# || 188.0 || VALID | | || 44 || #4PAB# || #4P9H# || 188.0 || VALID | ||
|- | |- | ||
− | | PL: RBS. | + | | PL: RBS.phi in pSB1A30 || 49 || #PEA6# || #TFE1# || 168.4 || VALID |
|- | |- | ||
| || 50 || #814A# || #QHB3# || 139.7 || discard | | || 50 || #814A# || #QHB3# || 139.7 || discard | ||
Line 432: | Line 433: | ||
| || 52 || #VBEZ# || #B9XO# || 135.2 || discard | | || 52 || #VBEZ# || #B9XO# || 135.2 || discard | ||
|- | |- | ||
− | | XL: RBS. | + | | XL: RBS.xpk in pSB1A30 || 57 || #4AE3# || #NF66# || 110.5 || VALID |
|- | |- | ||
| || 62 || #1NFT# || #ONK4# || 268.2 || VALID | | || 62 || #1NFT# || #ONK4# || 268.2 || VALID | ||
|- | |- | ||
− | | RBS. | + | | RBS.xpk.B0015 in pSB1A30 || 7 || #KANH# || #CNEK# || 110.1 || VALID |
|- | |- | ||
− | | RBS. | + | | RBS.phi.B0015 in pSB1A30 || 13 || #RX39# || #D8S8# || 85.0 || VALID |
|- | |- | ||
| || 19 || #3MNR# || #PZ6F# || 45.5 || discard | | || 19 || #3MNR# || #PZ6F# || 45.5 || discard | ||
Line 455: | Line 456: | ||
** list of items to take to iAMB: | ** list of items to take to iAMB: | ||
*** KAPA Mastermix | *** KAPA Mastermix | ||
− | *** | + | *** Primers |
*** Master plates | *** Master plates | ||
*** PCR strips | *** PCR strips |
Revision as of 09:16, 18 September 2015