Difference between revisions of "Team:Aachen/Notebook/Documentation/Methanol Monocistronic Diversity Library"

(→‎15-08-10)
(→‎15-08-20)
 
(11 intermediate revisions by the same user not shown)
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|}
 
|}
  
−
===Colony PCRs===
+
====Colony PCRs====
 
'''Program:'''
 
'''Program:'''
 
{|class="wikitable"
 
{|class="wikitable"
Line 247: Line 247:
 
! construct !! Primers !! elongation time !! expected length [bp] !! tested clones !! positive clones
 
! construct !! Primers !! elongation time !! expected length [bp] !! tested clones !! positive clones
 
|-
 
|-
−
| MDH.AP10 precursor || #MSP2#, #94LB# || 1'00'' || 920 || 1, 2, 3, 4, 5, 19, 20, 21 || -
+
| mdh.AP10 precursor || #MSP2#, #94LB# || 1'00'' || 920 || 1, 2, 3, 4, 5, 19, 20, 21 || -
 
|-
 
|-
−
| HPS.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 15
+
| hps.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 15
 
|-
 
|-
−
| HPS.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9
+
| hps.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9
 
|-
 
|-
−
| HPS.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-14 || 11, 13, 14
+
| hps.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-14 || 11, 13, 14
 
|-
 
|-
−
| HPS.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9, 13, 14 , 15, 16
+
| hps.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1160 || 9-16 || 9, 13, 14 , 15, 16
 
|-
 
|-
−
| PHI.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
+
| phi.AP00 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
 
|-
 
|-
−
| PHI.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
+
| phi.AP04 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || -
 
|-
 
|-
−
| PHI.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || 2, 3, 4, 10
+
| phi.AP10 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-8 || 2, 3, 4, 10
 
|-
 
|-
−
| PHI.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-6 || 5
+
| phi.AP19 precursor || #W6WP#, #94LB# || 1'13'' || 1074 || 1-6 || 5
−
|-
+
−
| AP19 polycistronic construct || varying || 2'15'' || varying || 0, 1, 3, 4 || 1
+
−
|-
+
−
|  || #W6WP#, #HSP2# ||  || 1878 || 0, 1, 3, 4 ||
+
−
|-
+
−
|  || #HSP1#, #XSP3# ||  || 1939 || 0, 1, 3, 4 ||
+
−
|-
+
−
|  || #XSP2#, #94LB# ||  || 2182 || 0, 1, 3, 4 ||
+
−
|-
+
−
| T7 polycistronic construct || varying || 2'15'' || varying || 1, 2, 7, 8 || -
+
−
|-
+
−
|  || #W6WP#, #HSP2# ||  || 1927 || 1, 2, 7, 8 ||
+
−
|-
+
−
|  || #HSP1#, #XSP3# ||  || 1939 || 1, 2, 7, 8 ||
+
−
|-
+
−
|  || #XSP2#, #94LB# ||  || 2182 || 1, 2, 7, 8 ||
+
 
|}
 
|}
−
* colony PCR of polycistronic constructs was repeated by Michael
 
  
−
===Assembly of MDH precursors===
+
====Assembly of MDH precursors====
−
* this time with blockers (find protocol in sciebo)
+
* this time with blockers; for higher coupling efficiency
−
**Assembly product MDH.AP00: #N1O1#
+
**Assembly product mdh.AP00: #N1O1#
−
**Assembly product MDH.AP04: #VYM6#
+
**Assembly product mdh.AP04: #VYM6#
−
**Assembly product MDH.AP10: #Q9QQ#
+
**Assembly product mdh.AP10: #Q9QQ#
−
**Assembly product MDH.AP19: #ONWS#
+
**Assembly product mdh.AP19: #ONWS#
  
 
==15-08-15==
 
==15-08-15==
−
===Tasks:===
+
====Tasks:====
−
* prep plasmid of yesterdays positive clones
+
* purify plasmid of prouven clones from  yesterday
−
* (repeat colony PCR of T7 polycistronic clones)
+
 
* '''make masterplates of MDH precursors (new assembly, this time with blockers)'''
 
* '''make masterplates of MDH precursors (new assembly, this time with blockers)'''
−
* assembly of HPS and PHI precursors (with blockers)
+
* assembly of hps and phi precursors (with blockers)
 
* tidy up fridge
 
* tidy up fridge
−
* plate trafos of HPS and PHI precursors  
+
* do transformation of hps and phi precursors  
−
* plate new trafos of MDH.AP04 and MDH.AP10 precursors
+
* do transformation of mdh.AP04 and mdh.AP10 precursors
−
* assemble MDH expression plasmid (FO4B.XYD9.PRDW.PLTB.OZ1D)
+
−
* trafo of MDH expression plasmid
+
−
* plate trafo on LB+K plates
+
  
−
===Plasmid prep of Hps and Phi precursors===
+
====Plasmid purification of hps and phi precursors====
  
 
{|class="wikitable"
 
{|class="wikitable"
 
! construct !! Cryo ID !! Plasmid ID !! Plasmid conc. [ng/µl]
 
! construct !! Cryo ID !! Plasmid ID !! Plasmid conc. [ng/µl]
 
|-
 
|-
−
| HPS AP19 #14 || #FOF4# || #B3PQ# || 170
+
| hps AP19 #14 || #FOF4# || #B3PQ# || 170
 
|-
 
|-
−
| HPS AP10 #14 || #638V# || #P4SB#|| 148
+
| hps AP10 #14 || #638V# || #P4SB#|| 148
 
|-
 
|-
−
| HPS AP10 #11 || #4ZSB# || #P89F#|| 143,5
+
| hps AP10 #11 || #4ZSB# || #P89F#|| 143,5
 
|-
 
|-
−
| HPS AP04 #9  || #NY1C# || #1TMT#|| 190,5
+
| hps AP04 #9  || #NY1C# || #1TMT#|| 190,5
 
|-
 
|-
−
| HPS AP19 #15 || #SM3L# || #NFOS#|| 204
+
| hps AP19 #15 || #SM3L# || #NFOS#|| 204
 
|-
 
|-
−
| HPS AP00 #15|| #HAKR# || #6ACO#|| 126,5
+
| hps AP00 #15|| #HAKR# || #6ACO#|| 126,5
 
|-
 
|-
−
| HPS AP19 #13 || #COMY# || #VSWC#|| 261
+
| hps AP19 #13 || #COMY# || #VSWC#|| 261
 
|-
 
|-
−
| HPS AP19 #9  || #OZ4O# || #L8Y4#|| 171,5
+
| hps AP19 #9  || #OZ4O# || #L8Y4#|| 171,5
 
|-
 
|-
−
| HPS AP10 #13 || #8P4S# || #4QLE#|| 238,5
+
| hps AP10 #13 || #8P4S# || #4QLE#|| 238,5
 
|-
 
|-
−
| HPS AP19 #16|| #CSLZ# || #6P68#|| 121,5
+
| hps AP19 #16|| #CSLZ# || #6P68#|| 121,5
 
|-
 
|-
−
| PHI AP10 #3 || #BV3E# || #L9E9#|| 161
+
| phi AP10 #3 || #BV3E# || #L9E9#|| 161
 
|-
 
|-
−
| PHI AP10 #4 || #OFHN# || #FVVW#|| 171,5
+
| phi AP10 #4 || #OFHN# || #FVVW#|| 171,5
 
|-
 
|-
−
| PHI AP19 #5 || #E6HX# || #HA1W#|| 192,5
+
| phi AP19 #5 || #E6HX# || #HA1W#|| 192,5
 
|}
 
|}
  
−
===Assembly of HPS precursors===
+
===Assembly of hps precursors===
−
* this time with blockers (find protocol in sciebo)
+
* this time with blockers (better coupling efficiency)
−
**Assembly product HPS.AP00: #WQPR#
+
**Assembly product hps.AP00: #WQPR#
−
**Assembly product HPS.AP04: #6WOE#
+
**Assembly product hps.AP04: #6WOE#
−
**Assembly product HPS.AP10: #XHXQ#
+
**Assembly product hps.AP10: #XHXQ#
−
**Assembly product HPS.AP19: #VM1V#
+
**Assembly product hps.AP19: #VM1V#
  
−
===Assembly of PHI precursors===
+
===Assembly of phi precursors===
−
* this time with blockers (find protocol in sciebo)
+
* this time with blockers (better coupling efficiency)
−
**Assembly product PHI.AP00: #AFA4#
+
**Assembly product phi.AP00: #AFA4#
−
**Assembly product PHI.AP04: #8FXO#
+
**Assembly product phi.AP04: #8FXO#
−
**Assembly product PHI.AP10: #KCDE#
+
**Assembly product phi.AP10: #KCDE#
−
**Assembly product PHI.AP19: #QHM6#
+
**Assembly product phi.AP19: #QHM6#
  
−
===Assembly of MDH expression plamid===
+
====Transformations====
−
* find protocol in sciebo
+
−
** assembly product AP19.MDH.TER in #DK1F#
+
−
 
+
−
===Transformations===
+
 
{|class="wikitable"
 
{|class="wikitable"
 
! # !! description !! assembly product  
 
! # !! description !! assembly product  
 
|-
 
|-
−
| 1 || HPS.AP00 precursor || #WQPR#  
+
| 1 || hps.AP00 precursor || #WQPR#  
 
|-
 
|-
−
| 2 || HPS.AP04 precursor || #6WOE#
+
| 2 || hps.AP04 precursor || #6WOE#
 
|-
 
|-
−
| 3 || HPS.AP10 precursor || #XHXQ#
+
| 3 || hps.AP10 precursor || #XHXQ#
 
|-
 
|-
−
| 4 || HPS.AP19 precursor || #VM1V#
+
| 4 || hps.AP19 precursor || #VM1V#
 
|-
 
|-
−
| 5 || PHI.AP00 precursor || #AFA4#
+
| 5 || phi.AP00 precursor || #AFA4#
 
|-
 
|-
−
| 6 || PHI.AP04 precursor || #8FXO#
+
| 6 || phi.AP04 precursor || #8FXO#
 
|-
 
|-
−
| 7 || PHI.AP10 precursor || #KCDE#
+
| 7 || phi.AP10 precursor || #KCDE#
 
|-
 
|-
−
| 8 || PHI.AP19 precursor || #QHM6#
+
| 8 || phi.AP19 precursor || #QHM6#
 
|-
 
|-
−
| 9 || MDH.AP04 precursor || #VYM6#
+
| 9 || mdh.AP04 precursor || #VYM6#
 
|-
 
|-
−
| 10 || MDH.AP10 precursor || #Q9QQ#
+
| 10 || mdh.AP10 precursor || #Q9QQ#
−
|-
+
−
| 11 || AP19.MDH.TER expression plasmid || #DK1F#
+
 
|}
 
|}
  
 
==15-08-16==
 
==15-08-16==
−
===Tasks:===
+
 
−
* prep plasmid of polycistronic clone #1
+
* make masterplates of yesterdays transformations
−
**Cryo #Q1Q3#
+
−
**purified plasmid #POLY#
+
−
* make masterplates of yesterdays trafos
+
−
* do/make colony PCR/overnights of yesterdays masterplates
+
−
* test KAPA 2G Mix
+
−
* do 'colony PCR' with purified plasmid of AP19 poly
+
  
 
==15-08-17==
 
==15-08-17==
Line 395: Line 362:
 
! construct !! cryo IDs !! plasmid IDs !! screening result  
 
! construct !! cryo IDs !! plasmid IDs !! screening result  
 
|-
 
|-
−
| MDH00 precursor || #L99Q#, #VLO3#, #EHQC#, #OD19#, #FHEL#, #MA9W# || #HR4E#, #ZZEE#, #3HPD#, #M1VB#, #QDVN#, #AHRM# || look at table below
+
| mdh00 precursor || #L99Q#, #VLO3#, #EHQC#, #OD19#, #FHEL#, #MA9W# || #HR4E#, #ZZEE#, #3HPD#, #M1VB#, #QDVN#, #AHRM# || results on table from tomorrow
 
|-
 
|-
−
| MDH04 precursor || #OB4C#, #TVND#, #1S1V#, #COXQ#, #3NZQ#, #YZXD# || #NEKS#, #OX44#, #P9HA#, #9CBA#, #SYMS#, #YY6O# ||
+
| mdh04 precursor || #OB4C#, #TVND#, #1S1V#, #COXQ#, #3NZQ#, #YZXD# || #NEKS#, #OX44#, #P9HA#, #9CBA#, #SYMS#, #YY6O# ||
 
|-
 
|-
−
| MDH19 precursor || #HBWZ#, #K3V6#, #CYF6#, #S1HT#, #TKO4#, #4MS8# || #BY9L#, #DNRY#, #KV18#, #1KEA#, #SPHL#, #N1BM# ||
+
| mdh19 precursor || #HBWZ#, #K3V6#, #CYF6#, #S1HT#, #TKO4#, #4MS8# || #BY9L#, #DNRY#, #KV18#, #1KEA#, #SPHL#, #N1BM# ||
 
|-
 
|-
−
| HPS00 precursor || #AVVO#, #X9PR#, #M4H8#, #44WP#, #DE4A#, #QMRB# || #XVYK#, #CMC1#, #ERZK#, #WXO8#, #D8PY#, #8AFD# ||
+
| hps00 precursor || #AVVO#, #X9PR#, #M4H8#, #44WP#, #DE4A#, #QMRB# || #XVYK#, #CMC1#, #ERZK#, #WXO8#, #D8PY#, #8AFD# ||
 
|-
 
|-
−
| HPS04 precursor || #8SW4#, #RSH9#, #1C8L#, #E1OQ#, #Y3TR#, #3BLV# || #YE8A#, #DERR#, #DVRP#, #FKNX#, #N3E4#, #HNV4# ||
+
| hps04 precursor || #8SW4#, #RSH9#, #1C8L#, #E1OQ#, #Y3TR#, #3BLV# || #YE8A#, #DERR#, #DVRP#, #FKNX#, #N3E4#, #HNV4# ||
 
|-
 
|-
−
| HPS10 precursor || #R8YT#, #QRZW#, #KVYA#, #NEFO#, #QN64#, #NSSM# || #1EQ3#, #PA13#, #FAES#, #Z64V#, #WSAD# ||
+
| hps10 precursor || #R8YT#, #QRZW#, #KVYA#, #NEFO#, #QN64#, #NSSM# || #1EQ3#, #PA13#, #FAES#, #Z64V#, #WSAD# ||
 
|-
 
|-
−
| PHI00 precursor || #TNBW#, #VSQN#, #YPFO#, #EELW#, #9H4T#, #VLHL# || #D6QY#, #931O#, #QWQP#, #AERW#, #ORX1#, #ZCTY# ||
+
| phi00 precursor || #TNBW#, #VSQN#, #YPFO#, #EELW#, #9H4T#, #VLHL# || #D6QY#, #931O#, #QWQP#, #AERW#, #ORX1#, #ZCTY# ||
 
|-
 
|-
−
| PHI04 precursor || #4MNP#, #DE96#, #YKZT#, #AQF8#, #ETTS#, #8COE# || #FXSC#, #P1O1#, #8CZD#, #EZYZ#, #VPP4#, #3EZP# ||
+
| phi04 precursor || #4MNP#, #DE96#, #YKZT#, #AQF8#, #ETTS#, #8COE# || #FXSC#, #P1O1#, #8CZD#, #EZYZ#, #VPP4#, #3EZP# ||
 
|-
 
|-
−
| PHI10 precursor || #SQ9N#, #AO6C#, #9RZL#, #48P3#, #PPWW#, #DB3R# || #FTX4#, #VM6Z#, #V4CA#, #XW1Y#, #AZND#, #WCF3# ||
+
| phi10 precursor || #SQ9N#, #AO6C#, #9RZL#, #48P3#, #PPWW#, #DB3R# || #FTX4#, #VM6Z#, #V4CA#, #XW1Y#, #AZND#, #WCF3# ||
 
|-
 
|-
−
| PHI19 precursor || #3WKA#, #94OY#, #TVM1#, #KPCQ#, #FB9L#, #HWM4# || #THQA#, #BND4#, #6H3Z#, #ME9O#, #39CF#, #RABS# ||
+
| phi19 precursor || #3WKA#, #94OY#, #TVM1#, #KPCQ#, #FB9L#, #HWM4# || #THQA#, #BND4#, #6H3Z#, #ME9O#, #39CF#, #RABS# ||
 
|}
 
|}
  
 
===further plasmids:===
 
===further plasmids:===
−
* AP19 Poly
 
−
** cryo: #LHZV#
 
−
** pasmid: #WVDR#
 
−
* MDH expression plasmid
 
−
** cryo: #MSX3#
 
−
** plasmid: #WDXZ#
 
 
* HPS19 precursor
 
* HPS19 precursor
 
** cryo: #ABCE#
 
** cryo: #ABCE#
 
** plasmid: #TTC4#
 
** plasmid: #TTC4#
−
 
−
'''AP19.MDH.Ter in pSB1KRDP'''
 
−
 
−
The MDH clones 2-19 were screened with a KAPA2G colony PCR. Controls are clone #1 and the plasmid #WDXZ#. Initial denaturation was set to 8'00" and the primer concentration was increased by 30 % to 6.5 mM. 0'15" denaturation and 0'15" annealing at 65-0.4°C per cycle. Elongation time was 0'20" for an expected product of 1205 bp (#W6WP# and #MSP3#). The PCR was not successful - only a band of plasmid template was visible.
 
−
 
−
Overnight cultures were prepared for all clones.
 
  
 
==15-08-18==
 
==15-08-18==
 
===Morning===
 
===Morning===
−
Plasmid preps and sequencing. All '''BOLD''' IDs do not exist yet. Prepping instructions:
+
Plasmid purifications and sequencing.  
−
* prep each of the 6 POLY cultures, but elute all into one target container named #C3NS#
+
−
* make '''cryos''' of the AP19.MDH cultures of clone #2+3 and then prep them. (both IDs in the table below)
+
−
 
+
 
Pipetting calculation table is ready at: '''\WetLab\Documentation\Methanol\15-08-18 Sequencings\'''
 
Pipetting calculation table is ready at: '''\WetLab\Documentation\Methanol\15-08-18 Sequencings\'''
  
Line 444: Line 396:
 
! construct & clone number !! cryo ID !! plasmid ID !! sequencing sample numbers !! sequencing result !! further procedure
 
! construct & clone number !! cryo ID !! plasmid ID !! sequencing sample numbers !! sequencing result !! further procedure
 
|-
 
|-
−
| AP19.POLY in pSB1KRDP #1 || #W3NB# || '''#C3NS#''' || 1-8 || NO CONTIGS FOUND || Repeat Sequencing
+
| mdh.TER.AP00 in pSB1CRDP #1 || #L99Q# || #HR4E# || 15 || MUTATION || Discard
 
|-
 
|-
−
| AP19.MDH.Ter in pSB1KRDP #1 || above || #WDXZ# || 9+10 || empty backbone || Discard
+
| mdh.TER.AP00 in pSB1CRDP #2 || #OD19# || #M1VB# || 16 || MUTATION || Discard
 
|-
 
|-
−
| AP19.MDH.Ter in pSB1KRDP #2 || '''#MSX3#''' || '''#FDX4#''' || 11+12 || mutation in promoter || Discard
+
| mdh.TER.AP00 in pSB1CRDP #6 || #MA9W# || #AHRM# || 17 || MUTATION || Discard
 
|-
 
|-
−
| AP19.MDH.Ter in pSB1KRDP #3 || '''#FZ8Q#''' || '''#MOMX#''' || 13+14 || empty backbone || Discard
+
| mdh.TER.AP04 in pSB1CRDP #1 || #OB4C# || #NEKS# || 18 || SPACER MUTATION || Discard
 
|-
 
|-
−
| MDH.TER.AP00 in pSB1CRDP #1 || #L99Q# || #HR4E# || 15 || MUTATION || Discard
+
| mdh.TER.AP04 in pSB1CRDP #2 || #TVND# || #OX44# || 19 || VALID || Keep
 
|-
 
|-
−
| MDH.TER.AP00 in pSB1CRDP #2 || #OD19# || #M1VB# || 16 || MUTATION || Discard
+
| mdh.TER.AP04 in pSB1CRDP #3 || #1S1V# || #P9HA# || 20 || VALID || Discard
 
|-
 
|-
−
| MDH.TER.AP00 in pSB1CRDP #6 || #MA9W# || #AHRM# || 17 || MUTATION || Discard
+
| mdh.TER.AP19 in pSB1CRDP #1 || #HBWZ# || #BY9L# || 21 || VALID || Discard
 
|-
 
|-
−
| MDH.TER.AP04 in pSB1CRDP #1 || #OB4C# || #NEKS# || 18 || SPACER MUTATION || Discard
+
| mdh.TER.AP19 in pSB1CRDP #2 || #K3V6# || #DNRY# || 22 || VALID || Discard
 
|-
 
|-
−
| MDH.TER.AP04 in pSB1CRDP #2 || #TVND# || #OX44# || 19 || VALID || Keep
+
| mdh.TER.AP19 in pSB1CRDP #3 || #CYF6# || #KV18# || 23 || VALID || Keep
 
|-
 
|-
−
| MDH.TER.AP04 in pSB1CRDP #3 || #1S1V# || #P9HA# || 20 || VALID || Discard
+
| hps.B1006.AP00 in pSB1CRDP #3 || #M4H8# || #ERZK# || 24 || VALID || Discard
 
|-
 
|-
−
| MDH.TER.AP19 in pSB1CRDP #1 || #HBWZ# || #BY9L# || 21 || VALID || Discard
+
| hps.B1006.AP00 in pSB1CRDP #4 || #44WP# || #WXO8# || 25 || VALID || Keep
 
|-
 
|-
−
| MDH.TER.AP19 in pSB1CRDP #2 || #K3V6# || #DNRY# || 22 || VALID || Discard
+
| hps.B1006.AP00 in pSB1CRDP #5 || #DE4A# || #D8PY# || 26 || NO CONTIGS FOUND || Discard
 
|-
 
|-
−
| MDH.TER.AP19 in pSB1CRDP #3 || #CYF6# || #KV18# || 23 || VALID || Keep
+
| hps.B1006.AP04 in pSB1CRDP #2 || #RSH9# || #DERR# || 27 || MUTATION || Discard
 
|-
 
|-
−
| HPS.B1006.AP00 in pSB1CRDP #3 || #M4H8# || #ERZK# || 24 || VALID || Discard
+
| hps.B1006.AP04 in pSB1CRDP #3 || #1C8L# || #DVRP# || 28 || MUTATION || Discard
 
|-
 
|-
−
| HPS.B1006.AP00 in pSB1CRDP #4 || #44WP# || #WXO8# || 25 || VALID || Keep
+
| hps.B1006.AP04 in pSB1CRDP #6 || #3BLV# || #HNV4# || 29 || MUTATION || Discard
 
|-
 
|-
−
| HPS.B1006.AP00 in pSB1CRDP #5 || #DE4A# || #D8PY# || 26 || NO CONTIGS FOUND || Discard
+
| hps.B1006.AP10 in pSB1CRDP #2 || #QRZW# || #1EQ3# || 30 || NO CONTIGS FOUND || Discard
−
|-
+
−
| HPS.B1006.AP04 in pSB1CRDP #2 || #RSH9# || #DERR# || 27 || MUTATION || Discard
+
−
|-
+
−
| HPS.B1006.AP04 in pSB1CRDP #3 || #1C8L# || #DVRP# || 28 || MUTATION || Discard
+
−
|-
+
−
| HPS.B1006.AP04 in pSB1CRDP #6 || #3BLV# || #HNV4# || 29 || MUTATION || Discard
+
−
|-
+
−
| HPS.B1006.AP10 in pSB1CRDP #2 || #QRZW# || #1EQ3# || 30 || NO CONTIGS FOUND || Discard
+
 
|-  
 
|-  
−
| HPS.B1006.AP10 in pSB1CRDP #4 || #NEFO# || #FAES# || 31 || NO CONTIGS FOUND|| Discard
+
| hps.B1006.AP10 in pSB1CRDP #4 || #NEFO# || #FAES# || 31 || NO CONTIGS FOUND|| Discard
 
|-
 
|-
−
| HPS.B1006.AP19 in pSB1CRDP #1 || #ABCE# || #TTC4# || 32 || MUTATION || Discard
+
| hps.B1006.AP19 in pSB1CRDP #1 || #ABCE# || #TTC4# || 32 || MUTATION || Discard
 
|-
 
|-
−
| PHI.B1002.AP00 in pSB1CRDP #2 || #VSQN# || #931O# || 33 || VALID || Keep
+
| phi.B1002.AP00 in pSB1CRDP #2 || #VSQN# || #931O# || 33 || VALID || Keep
 
|-
 
|-
−
| PHI.B1002.AP00 in pSB1CRDP #3 || #YPFO# || #QWQP# || 34 || NO CONTIGS FOUND || Discard
+
| phi.B1002.AP00 in pSB1CRDP #3 || #YPFO# || #QWQP# || 34 || NO CONTIGS FOUND || Discard
 
|-
 
|-
−
| PHI.B1002.AP00 in pSB1CRDP #4 || #EELW# || #AERW# || 35 || MUTATION || Discard
+
| phi.B1002.AP00 in pSB1CRDP #4 || #EELW# || #AERW# || 35 || MUTATION || Discard
 
|-
 
|-
−
| PHI.B1002.AP04 in pSB1CRDP #1 || #4MNP# || #FXSC# || 36 || NO CONTIGS FOUND || Discard
+
| phi.B1002.AP04 in pSB1CRDP #1 || #4MNP# || #FXSC# || 36 || NO CONTIGS FOUND || Discard
 
|-
 
|-
−
| PHI.B1002.AP04 in pSB1CRDP #3 || #YKZT# || #8CZD# || 37 || PROMOTER OK || Discard
+
| phi.B1002.AP04 in pSB1CRDP #3 || #YKZT# || #8CZD# || 37 || PROMOTER OK || Discard
 
|-
 
|-
−
| PHI.B1002.AP04 in pSB1CRDP #4 || #AQF8# || #EZYZ# || 38 || VALID || Keep
+
| phi.B1002.AP04 in pSB1CRDP #4 || #AQF8# || #EZYZ# || 38 || VALID || Keep
 
|-
 
|-
−
| PHI.B1002.AP10 in pSB1CRDP #3 || #9RZL# || #V4CA# || 39 || VALID || Keep
+
| phi.B1002.AP10 in pSB1CRDP #3 || #9RZL# || #V4CA# || 39 || VALID || Keep
 
|-
 
|-
−
| PHI.B1002.AP10 in pSB1CRDP #4 || #48P3# || #XW1Y# || 40 || VALID || Discard
+
| phi.B1002.AP10 in pSB1CRDP #4 || #48P3# || #XW1Y# || 40 || VALID || Discard
 
|-
 
|-
−
| PHI.B1002.AP10 in pSB1CRDP #6 || #DB3R# || #WCF3# || 41 || VALID || Discard
+
| phi.B1002.AP10 in pSB1CRDP #6 || #DB3R# || #WCF3# || 41 || VALID || Discard
 
|-
 
|-
−
| PHI.B1002.AP19 in pSB1CRDP #2 || #94OY# || #BND4# || 42 || NO CONTIGS FOUND || Discard
+
| phi.B1002.AP19 in pSB1CRDP #2 || #94OY# || #BND4# || 42 || NO CONTIGS FOUND || Discard
 
|-
 
|-
−
| PHI.B1002.AP19 in pSB1CRDP #5 || #FB9L# || #39CF# || 43 || MUTATION || Discard
+
| phi.B1002.AP19 in pSB1CRDP #5 || #FB9L# || #39CF# || 43 || MUTATION || Discard
 
|-
 
|-
−
| PHI.B1002.AP19 in pSB1CRDP #6 || #HWM4# || #RABS# || 44 || VALID || Keep
+
| phi.B1002.AP19 in pSB1CRDP #6 || #HWM4# || #RABS# || 44 || VALID || Keep
 
|}
 
|}
  
−
===Noon===
+
 
−
Prep plasmids of T7.POLY in BL21 (DE3)
+
==15-08-20==
 +
===missing Precursors:===
 +
 
 +
do cryo and plasmidprep of HPS AP04  #7-14 & HPS AP10 #7-14 & MDH AP00  #7-14
  
 
{|class="wikitable"
 
{|class="wikitable"
−
! construct & clone number !! cryo ID !! plasmid ID !! check PCR result
+
! construct & clone number !! cryo ID !! plasmid ID !! further procedure
 
|-
 
|-
−
| T7.lacO.POLY in pSB1KRDP #1 || #LHBN# || #VYCO# || discard
+
| HPS.B1006.AP04 in pSB1CRDP #7 || #RBVV# || #C4VV#   || cancel
 
|-
 
|-
−
| T7.lacO.POLY in pSB1KRDP #5 || #HB93# || #ANPV# || discard
+
| HPS.B1006.AP04 in pSB1CRDP #8 || #LACA# || #FCOY# || cancel
 
|-
 
|-
−
| T7.lacO.POLY in pSB1KRDP #6 || #6LOZ# || #96TS# || discard
+
| HPS.B1006.AP04 in pSB1CRDP #9 || #E9VF# || #ZNWW# || cancel
 
|-
 
|-
−
| T7.lacO.POLY in pSB1KRDP #7 || #KYOQ# || #XVE3# || discard
+
| HPS.B1006.AP04 in pSB1CRDP #10 || #DYDR# || #O3AH# || cancel
 
|-
 
|-
−
| T7.lacO.POLY in pSB1KRDP #8 || #MCMR# || #BSLA# || discard
+
| HPS.B1006.AP04 in pSB1CRDP #11 || #DRNL# || #MVSO# || cancel
−
|}
+
−
 
+
−
* RDP-Assembly of T7.lacO.MDH.Ter in pSB1KRDP (product: #....#)
+
−
* transform #....# into BL21 Gold (DE3) AND DH5a
+
−
====Check-PCR the prepped T7.POLY plasmids====
+
−
*with the three primer pairs, to find a complete construct
+
−
*elongation time 0'33''
+
−
* #POLY# as positive control (fitting bands already 15-08-16)
+
−
{|class="wikitable"
+
−
! Part ID !! Sample !! Primers !! expected length [bp] !! result
+
 
|-
 
|-
−
| #VYCO# || 1 || #W6WP# + #HSP2# || 1927 || negative
+
| HPS.B1006.AP04 in pSB1CRDP #12 || #MFPT# || #VOLC# || cancel
 
|-
 
|-
−
| || 2 || #HSP1# + #XSP3#|| 1939 || negative
+
| HPS.B1006.AP04 in pSB1CRDP #13 || #SL4H# || #YNXS# || cancel
 
|-
 
|-
−
| || 3 || #XSP2# + #94LB#|| 2182 || negative
+
| HPS.B1006.AP04 in pSB1CRDP #14 || #KNX8# || #XOAK# || cancel
 
|-
 
|-
−
| #ANPV# || 4 || #W6WP# + #HSP2#||1927 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #7 || #ABAN# || #FMV3# || cancel
 
|-
 
|-
−
| || 5 || #HSP1# + #XSP3#||1939 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #8 || #N1RL# || #QPAO# || cancel
 
|-
 
|-
−
| || 6 || #XSP2# + #94LB#||2182 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #9 || #3C6P# || #BRAM# || cancel
 
|-
 
|-
−
| #96TS# || 7 || #W6WP# + #HSP2#||1927 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #10 || #TDYL# || #T4FR# || cancel
 
|-
 
|-
−
| || 8 || #HSP1# + #XSP3#||1939 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #11 || #1WXS# || #O4K8# || cancel
 
|-
 
|-
−
| || 9 || #XSP2# + #94LB#||2182 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #12 || #OTE8# || #HRWD#   || cancel
 
|-
 
|-
−
| #XVE3# || 10 || #W6WP# + #HSP2#||1927 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #13 || #1FX9# || #QZCX# || cancel
 
|-
 
|-
−
| || 11 || #HSP1# + #XSP3#||1939 || negative
+
| HPS.B1006.AP10 in pSB1CRDP #14 || #86CB# || #YBTE#   || cancel
 
|-
 
|-
−
| || 12 || #XSP2# + #94LB#||2182 || negative
+
| MDH.TER.AP00 in pSB1CRDP #7 || #3QEE# || #TDLS# || cancel
 
|-
 
|-
−
| #BSLA# || 13 || #W6WP# + #HSP2#||1927 || negative
+
| MDH.TER.AP00 in pSB1CRDP #8 || #RFK6# || #8E93#   || cancel
 
|-
 
|-
−
| || 14 || #HSP1# + #XSP3#||1939 || negative
+
| MDH.TER.AP00 in pSB1CRDP #9 || #NDM6# || #F69B# || cancel
 
|-
 
|-
−
| || 15 || #XSP2# + #94LB#||2182 || negative
+
| MDH.TER.AP00 in pSB1CRDP #10 || #PR4Z# || #FN8P# || cancel
 
|-
 
|-
−
| #POLY# || 16 || #W6WP# + #HSP2#||1927 || positive
+
| MDH.TER.AP00 in pSB1CRDP #11 || #RAYK# || #YF83# || cancel
 
|-
 
|-
−
| || 17 || #HSP1# + #XSP3#||1939 || positive
+
| MDH.TER.AP00 in pSB1CRDP #12 || #HQKY# || #MFCB# || cancel
 
|-
 
|-
−
| || 18 || #XSP2# + #94LB#||2182 || positive
+
| MDH.TER.AP00 in pSB1CRDP #13 || #R1YY# || #XFLS# || cancel
 +
|-
 +
| MDH.TER.AP00 in pSB1CRDP #14 || #CF4X# || #1131#  || cancel
 
|}
 
|}
−
* Label yesterdays SDS gel and upload the results to the internal wiki.
 
  
−
===Afternoon===
+
*as problems seem to be mutations in the promoters, building precursors project will be canceled
−
* make M9+K+Glucose40mM overnight cultures of
+
*we don't have time and money to order new HPLC purified promoters
−
** B0015 in pSB1K3 in DH5a
+
−
** AP19.POLY in pSB1KRDP in DH5a clone #1
+
−
** AP19.MDH.Ter in pSB1KRDP in DH5a clone #1-6
+
−
* make two LB+K overnight cultures of T7.POLY on LB+K
+
−
 
+
−
====RDP Assembly====
+
−
* Assembly of #FO4B# and #PVFL# (Kanamycin Cap with high copy cap) the product is #QWRC# -> we need this strain as a negative control that carries the plasmid but does not express anything
+
−
 
+
−
Transform important plasmids into BL21 Gold (DE3) and plate on M9+K+40mMGlucose as well as LB+K
+
−
{|class="wikitable"
+
−
! construct !! transformed plasmid !! BL21 Gold (DE3) cryo
+
−
|-
+
−
| AP19.POLY in pSB1KRDP || #POLY# || #....#
+
−
|-
+
−
| AP19.MDH.Ter in pSB1KRDP || #WDXZ#, #FDX4#, #MOMX# || #....#
+
−
|-
+
−
| pSB1KRDP || #QWRC#  || #....#
+
−
|}
+
  
 
=References=
 
=References=

Latest revision as of 12:53, 18 September 2015


Laboratory Notebook

15-08-08

Constructing Precursor Plasmids

gene RDP parts product in expected length / result plasmid ID cryo ID
Mdh (mdh.TER.AP00) #XOXM# + #PRDW# + #PLTB# + #PQT6# + #PVFL# #K3YV# ; empty 3497 bp / no evaluable result #CDPA#, #ZYBC# #SF63#, #MBRR#
Mdh (mdh.TER.AP04) #XOXM# + #PRDW# + #PLTB# + #WPW6# + #PVFL# #AVQ6#; empty 3497 bp / no evaluable result #YNOB#, #1KSW# #DCN9#, #WHBF#
Mdh (mdh.TER.AP10) #XOXM# + #PRDW# + #PLTB# + #3S8V# + #PVFL# #ZE1A#; empty 3497 bp / no evaluable result #EBWO#, #936C# #8KAA#, #EB88#
Mdh (mdh.TER.AP19) #XOXM# + #PRDW# + #PLTB# + #MBWS# + #PVFL# #NDKS#; empty 3497 bp / no evaluable result #1MC6#, #ESQD# #48C8#, #8BZ9#
  • heat shocked in DH5alpha with 5 µl of DNA

15-08-09

Gel control of mdh precursor plasmids:

  • no good picture could be taken because the UV light destroyed the DNA; picture of it will be taken tomorrow
  • plasmid purification and cryo cultures of mdh precursor plasmids

15-08-10

  • make cryo cultures and purify plasmids of mdh precursor plasmids
  • Measure concentrations of purified mdh precursor plasmids
  • Gel control of mdh precursor plasmids
  • Pipett sequencing of mdh precursor plasmids
  • test digest of mdh precursor plasmids:
digest ID template ID expected lengths [bp] result
A #CDPA# 2226 / 1279 2226 / -
B #ZYBC# 2226 / 1279 2226 / 1700
C #YNOB# 2226 / 1279 2226 / 1700
D #1KSW# 2226 / 1279 2226 / 1700
E #EBWO# 2226 / 1279 2226 / -
F #936C# 2226 / 1279 2226 / -
G #1MC6# 2226 / 1279 2226 / 1279
H #ESQD# 2226 / 1279 2226 / 1279

digests stored in falcon #PSHF#

15-08-11

  • sequencing of following samples: #1MC6#, #ESQD#,
  • oligo assembly: BBa_B1002-RDP (#HTSR#), BBa_B1006-RDP (#8AMS#), AP19-RDP (#MBWS#)
    • heated on 90 °C, then linear cool down in 45 minutes to room temperature

assembly of hps Precursors

gene RDP parts product in
hps (hps.B1006.AP00) #RYPQ# + #VO4C# + #8AMS# + #M1ZY# + #OZD1# #V9H9#
hps (hps.B1006.AP04) #RYPQ# + #VO4C# + #8AMS# + #HB96# + #OZD1# #XKX6#
hps (hps.B1006.AP10) #RYPQ# + #VO4C# + #8AMS# + #4T9A# + #OZD1# #SNDK#
hps (hps.B1006.AP19) #RYPQ# + #VO4C# + #8AMS# + #XYD9# + #OZD1# #BC4V#


  • heat shock transformation in DH5alpha with 5 µl of DNA

15-08-12

  • there were colonies on the transformation plates of the hps precursors
  • Oligos from 15-08-11: put them on 2% agarose:

(good results: 0,8µl DNA and more than 3,5 µl 50bp Ladder)

part expected lenght result
BBa_B1002-RDP (#HTSR#) 45 bp fragments of ca. 50bp
BBa_B1006-RDP (#8AMS#) 50 bp fragments of ca. 50bp
AP19-RDP(#MBWS#) 47 bp fragments of ca. 50bp

Assembly of phi precursors

gene RDP parts product in
phi (phi.B1002.AP00) #VVSC#
phi (phi.B1002.AP04) #BNHW#
phi (phi.B1002.AP10) #D1LL#
phi (phi.B1002.AP19) #EB8T#
  • each 5µl transformed in DH5alpha
  • masterplates and colony PCR will follow

colony PCR of mdh precursor clones

  • primers: #MSP2# and #94LB#
  • elongation time: 1'00
  • annealing temperature: 58°C
  • expected length: 920 bp
ID description positive clones
A 1-12 mdh.Ter.AP00 no
B 1-15 mdh.Ter.AP04 5
C 1-12 mdh.Ter.AP10 no
D 1-11 mdh.Ter.AP19 2, 4

15-08-13

tasks:

  • colony PCR of hps precursors
  • colony PCR of mdh precursors (again)
  • masterplates of phi precursors
  • overnight cultures of positive colony PCR clones

Colony PCRs

mdh precursor colony PCR:

  • Primers: #MSP2#, #94LB#
step time Temp [°C]
denature 5'00 94
denature 0'30 94
anneal 0'30 55
elongate 1'00 68
elongate 2'00 68
store forever 16

hps precursor colony PCR:

  • Primers: #HSP1#, #94LB#
step time Temp [°C]
denature 5'00 94
denature 0'30 94
anneal 0'30 55
elongate 0'40 68
elongate 2'00 68
store forever 16

Results:

construct ID construct description expected length [bp] positive clones
A 1-8 mdh AP00 precursor 920 bp (A16), A18
B 1-8 mdh AP04 precursor 920 bp B15, (B17), (B18)
C 1-8 mdh AP10 precursor 920 bp -
D 1-8 mdh AP19 precursor 920 bp D12, D14
E 1-8 hps AP00 precursor 579 bp -
F 1-8 hps AP04 precursor 579 bp -
G 1-8 hps AP10 precursor 579 bp -
H 1-8 hps AP19 precursor 579 bp -

15-08-14

tasks:

  • colony PCR of all phi precursors
  • colony PCR of all hps precursors
  • colony PCR of mdh AP10 precursor
  • purify plasmid of:
    • mdh AP00 precursors (A16, A18)
    • mdh AP04 precursors (B15, B17, B18)
    • mdh AP19 precursors (D12, D14)
  • label gels of yesterday
  • BsaI digest clean-up

purified plasmids and cryo cultures of mdh precursors

construct Cryo ID Plasmid ID Plasmid conc. [ng/µl]
mdh AP00 #16 #Q6XM# #CZ83# 125,5
mdh AP00 #18 #6F1N# #XNTZ# 171,5
mdh AP04 #15 #848Y# #SRYN# 338
mdh AP04 #17 #FCKK# #TK9X# 134
mdh AP04 #18 #3C1N# #C8EB# 147,5
mdh AP19 #12 #XYM1# #VMBV# 72
mdh AP19 #14 #RB99# #4P6A# 101,5

Colony PCRs

Program:

step time T [°C]
denature 5'00 94
denature 0'30 94
annealing 0'30 55
elongation varying 68
final elongation 2'00 68
store forever 16

Results table:

construct Primers elongation time expected length [bp] tested clones positive clones
mdh.AP10 precursor #MSP2#, #94LB# 1'00 920 1, 2, 3, 4, 5, 19, 20, 21 -
hps.AP00 precursor #W6WP#, #94LB# 1'13 1160 9-16 15
hps.AP04 precursor #W6WP#, #94LB# 1'13 1160 9-16 9
hps.AP10 precursor #W6WP#, #94LB# 1'13 1160 9-14 11, 13, 14
hps.AP19 precursor #W6WP#, #94LB# 1'13 1160 9-16 9, 13, 14 , 15, 16
phi.AP00 precursor #W6WP#, #94LB# 1'13 1074 1-8 -
phi.AP04 precursor #W6WP#, #94LB# 1'13 1074 1-8 -
phi.AP10 precursor #W6WP#, #94LB# 1'13 1074 1-8 2, 3, 4, 10
phi.AP19 precursor #W6WP#, #94LB# 1'13 1074 1-6 5

Assembly of MDH precursors

  • this time with blockers; for higher coupling efficiency
    • Assembly product mdh.AP00: #N1O1#
    • Assembly product mdh.AP04: #VYM6#
    • Assembly product mdh.AP10: #Q9QQ#
    • Assembly product mdh.AP19: #ONWS#

15-08-15

Tasks:

  • purify plasmid of prouven clones from yesterday
  • make masterplates of MDH precursors (new assembly, this time with blockers)
  • assembly of hps and phi precursors (with blockers)
  • tidy up fridge
  • do transformation of hps and phi precursors
  • do transformation of mdh.AP04 and mdh.AP10 precursors

Plasmid purification of hps and phi precursors

construct Cryo ID Plasmid ID Plasmid conc. [ng/µl]
hps AP19 #14 #FOF4# #B3PQ# 170
hps AP10 #14 #638V# #P4SB# 148
hps AP10 #11 #4ZSB# #P89F# 143,5
hps AP04 #9 #NY1C# #1TMT# 190,5
hps AP19 #15 #SM3L# #NFOS# 204
hps AP00 #15 #HAKR# #6ACO# 126,5
hps AP19 #13 #COMY# #VSWC# 261
hps AP19 #9 #OZ4O# #L8Y4# 171,5
hps AP10 #13 #8P4S# #4QLE# 238,5
hps AP19 #16 #CSLZ# #6P68# 121,5
phi AP10 #3 #BV3E# #L9E9# 161
phi AP10 #4 #OFHN# #FVVW# 171,5
phi AP19 #5 #E6HX# #HA1W# 192,5

Assembly of hps precursors

  • this time with blockers (better coupling efficiency)
    • Assembly product hps.AP00: #WQPR#
    • Assembly product hps.AP04: #6WOE#
    • Assembly product hps.AP10: #XHXQ#
    • Assembly product hps.AP19: #VM1V#

Assembly of phi precursors

  • this time with blockers (better coupling efficiency)
    • Assembly product phi.AP00: #AFA4#
    • Assembly product phi.AP04: #8FXO#
    • Assembly product phi.AP10: #KCDE#
    • Assembly product phi.AP19: #QHM6#

Transformations

# description assembly product
1 hps.AP00 precursor #WQPR#
2 hps.AP04 precursor #6WOE#
3 hps.AP10 precursor #XHXQ#
4 hps.AP19 precursor #VM1V#
5 phi.AP00 precursor #AFA4#
6 phi.AP04 precursor #8FXO#
7 phi.AP10 precursor #KCDE#
8 phi.AP19 precursor #QHM6#
9 mdh.AP04 precursor #VYM6#
10 mdh.AP10 precursor #Q9QQ#

15-08-16

  • make masterplates of yesterdays transformations

15-08-17

Precursor plasmids:

construct cryo IDs plasmid IDs screening result
mdh00 precursor #L99Q#, #VLO3#, #EHQC#, #OD19#, #FHEL#, #MA9W# #HR4E#, #ZZEE#, #3HPD#, #M1VB#, #QDVN#, #AHRM# results on table from tomorrow
mdh04 precursor #OB4C#, #TVND#, #1S1V#, #COXQ#, #3NZQ#, #YZXD# #NEKS#, #OX44#, #P9HA#, #9CBA#, #SYMS#, #YY6O#
mdh19 precursor #HBWZ#, #K3V6#, #CYF6#, #S1HT#, #TKO4#, #4MS8# #BY9L#, #DNRY#, #KV18#, #1KEA#, #SPHL#, #N1BM#
hps00 precursor #AVVO#, #X9PR#, #M4H8#, #44WP#, #DE4A#, #QMRB# #XVYK#, #CMC1#, #ERZK#, #WXO8#, #D8PY#, #8AFD#
hps04 precursor #8SW4#, #RSH9#, #1C8L#, #E1OQ#, #Y3TR#, #3BLV# #YE8A#, #DERR#, #DVRP#, #FKNX#, #N3E4#, #HNV4#
hps10 precursor #R8YT#, #QRZW#, #KVYA#, #NEFO#, #QN64#, #NSSM# #1EQ3#, #PA13#, #FAES#, #Z64V#, #WSAD#
phi00 precursor #TNBW#, #VSQN#, #YPFO#, #EELW#, #9H4T#, #VLHL# #D6QY#, #931O#, #QWQP#, #AERW#, #ORX1#, #ZCTY#
phi04 precursor #4MNP#, #DE96#, #YKZT#, #AQF8#, #ETTS#, #8COE# #FXSC#, #P1O1#, #8CZD#, #EZYZ#, #VPP4#, #3EZP#
phi10 precursor #SQ9N#, #AO6C#, #9RZL#, #48P3#, #PPWW#, #DB3R# #FTX4#, #VM6Z#, #V4CA#, #XW1Y#, #AZND#, #WCF3#
phi19 precursor #3WKA#, #94OY#, #TVM1#, #KPCQ#, #FB9L#, #HWM4# #THQA#, #BND4#, #6H3Z#, #ME9O#, #39CF#, #RABS#

further plasmids:

  • HPS19 precursor
    • cryo: #ABCE#
    • plasmid: #TTC4#

15-08-18

Morning

Plasmid purifications and sequencing. Pipetting calculation table is ready at: \WetLab\Documentation\Methanol\15-08-18 Sequencings\

construct & clone number cryo ID plasmid ID sequencing sample numbers sequencing result further procedure
mdh.TER.AP00 in pSB1CRDP #1 #L99Q# #HR4E# 15 MUTATION Discard
mdh.TER.AP00 in pSB1CRDP #2 #OD19# #M1VB# 16 MUTATION Discard
mdh.TER.AP00 in pSB1CRDP #6 #MA9W# #AHRM# 17 MUTATION Discard
mdh.TER.AP04 in pSB1CRDP #1 #OB4C# #NEKS# 18 SPACER MUTATION Discard
mdh.TER.AP04 in pSB1CRDP #2 #TVND# #OX44# 19 VALID Keep
mdh.TER.AP04 in pSB1CRDP #3 #1S1V# #P9HA# 20 VALID Discard
mdh.TER.AP19 in pSB1CRDP #1 #HBWZ# #BY9L# 21 VALID Discard
mdh.TER.AP19 in pSB1CRDP #2 #K3V6# #DNRY# 22 VALID Discard
mdh.TER.AP19 in pSB1CRDP #3 #CYF6# #KV18# 23 VALID Keep
hps.B1006.AP00 in pSB1CRDP #3 #M4H8# #ERZK# 24 VALID Discard
hps.B1006.AP00 in pSB1CRDP #4 #44WP# #WXO8# 25 VALID Keep
hps.B1006.AP00 in pSB1CRDP #5 #DE4A# #D8PY# 26 NO CONTIGS FOUND Discard
hps.B1006.AP04 in pSB1CRDP #2 #RSH9# #DERR# 27 MUTATION Discard
hps.B1006.AP04 in pSB1CRDP #3 #1C8L# #DVRP# 28 MUTATION Discard
hps.B1006.AP04 in pSB1CRDP #6 #3BLV# #HNV4# 29 MUTATION Discard
hps.B1006.AP10 in pSB1CRDP #2 #QRZW# #1EQ3# 30 NO CONTIGS FOUND Discard
hps.B1006.AP10 in pSB1CRDP #4 #NEFO# #FAES# 31 NO CONTIGS FOUND Discard
hps.B1006.AP19 in pSB1CRDP #1 #ABCE# #TTC4# 32 MUTATION Discard
phi.B1002.AP00 in pSB1CRDP #2 #VSQN# #931O# 33 VALID Keep
phi.B1002.AP00 in pSB1CRDP #3 #YPFO# #QWQP# 34 NO CONTIGS FOUND Discard
phi.B1002.AP00 in pSB1CRDP #4 #EELW# #AERW# 35 MUTATION Discard
phi.B1002.AP04 in pSB1CRDP #1 #4MNP# #FXSC# 36 NO CONTIGS FOUND Discard
phi.B1002.AP04 in pSB1CRDP #3 #YKZT# #8CZD# 37 PROMOTER OK Discard
phi.B1002.AP04 in pSB1CRDP #4 #AQF8# #EZYZ# 38 VALID Keep
phi.B1002.AP10 in pSB1CRDP #3 #9RZL# #V4CA# 39 VALID Keep
phi.B1002.AP10 in pSB1CRDP #4 #48P3# #XW1Y# 40 VALID Discard
phi.B1002.AP10 in pSB1CRDP #6 #DB3R# #WCF3# 41 VALID Discard
phi.B1002.AP19 in pSB1CRDP #2 #94OY# #BND4# 42 NO CONTIGS FOUND Discard
phi.B1002.AP19 in pSB1CRDP #5 #FB9L# #39CF# 43 MUTATION Discard
phi.B1002.AP19 in pSB1CRDP #6 #HWM4# #RABS# 44 VALID Keep


15-08-20

missing Precursors:

do cryo and plasmidprep of HPS AP04 #7-14 & HPS AP10 #7-14 & MDH AP00 #7-14

construct & clone number cryo ID plasmid ID further procedure
HPS.B1006.AP04 in pSB1CRDP #7 #RBVV# #C4VV# cancel
HPS.B1006.AP04 in pSB1CRDP #8 #LACA# #FCOY# cancel
HPS.B1006.AP04 in pSB1CRDP #9 #E9VF# #ZNWW# cancel
HPS.B1006.AP04 in pSB1CRDP #10 #DYDR# #O3AH# cancel
HPS.B1006.AP04 in pSB1CRDP #11 #DRNL# #MVSO# cancel
HPS.B1006.AP04 in pSB1CRDP #12 #MFPT# #VOLC# cancel
HPS.B1006.AP04 in pSB1CRDP #13 #SL4H# #YNXS# cancel
HPS.B1006.AP04 in pSB1CRDP #14 #KNX8# #XOAK# cancel
HPS.B1006.AP10 in pSB1CRDP #7 #ABAN# #FMV3# cancel
HPS.B1006.AP10 in pSB1CRDP #8 #N1RL# #QPAO# cancel
HPS.B1006.AP10 in pSB1CRDP #9 #3C6P# #BRAM# cancel
HPS.B1006.AP10 in pSB1CRDP #10 #TDYL# #T4FR# cancel
HPS.B1006.AP10 in pSB1CRDP #11 #1WXS# #O4K8# cancel
HPS.B1006.AP10 in pSB1CRDP #12 #OTE8# #HRWD# cancel
HPS.B1006.AP10 in pSB1CRDP #13 #1FX9# #QZCX# cancel
HPS.B1006.AP10 in pSB1CRDP #14 #86CB# #YBTE# cancel
MDH.TER.AP00 in pSB1CRDP #7 #3QEE# #TDLS# cancel
MDH.TER.AP00 in pSB1CRDP #8 #RFK6# #8E93# cancel
MDH.TER.AP00 in pSB1CRDP #9 #NDM6# #F69B# cancel
MDH.TER.AP00 in pSB1CRDP #10 #PR4Z# #FN8P# cancel
MDH.TER.AP00 in pSB1CRDP #11 #RAYK# #YF83# cancel
MDH.TER.AP00 in pSB1CRDP #12 #HQKY# #MFCB# cancel
MDH.TER.AP00 in pSB1CRDP #13 #R1YY# #XFLS# cancel
MDH.TER.AP00 in pSB1CRDP #14 #CF4X# #1131# cancel
  • as problems seem to be mutations in the promoters, building precursors project will be canceled
  • we don't have time and money to order new HPLC purified promoters

References

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