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|
Line 390: |
Line 390: |
| ==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\''' |
| | | |
| {|class="wikitable" | | {|class="wikitable" |
| ! 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
| |
− | |-
| |
− | | AP19.MDH.Ter in pSB1KRDP #1 || above || #WDXZ# || 9+10 || empty backbone || Discard
| |
− | |-
| |
− | | AP19.MDH.Ter in pSB1KRDP #2 || '''#MSX3#''' || '''#FDX4#''' || 11+12 || mutation in promoter || Discard
| |
| |- | | |- |
| | AP19.MDH.Ter in pSB1KRDP #3 || '''#FZ8Q#''' || '''#MOMX#''' || 13+14 || empty backbone || Discard | | | AP19.MDH.Ter in pSB1KRDP #3 || '''#FZ8Q#''' || '''#MOMX#''' || 13+14 || empty backbone || Discard |
| |- | | |- |
− | | MDH.TER.AP00 in pSB1CRDP #1 || #L99Q# || #HR4E# || 15 || MUTATION || Discard | + | | 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 #2 || #OD19# || #M1VB# || 16 || MUTATION || Discard |
| |- | | |- |
− | | MDH.TER.AP00 in pSB1CRDP #6 || #MA9W# || #AHRM# || 17 || 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 #1 || #OB4C# || #NEKS# || 18 || SPACER MUTATION || Discard |
| |- | | |- |
− | | MDH.TER.AP04 in pSB1CRDP #2 || #TVND# || #OX44# || 19 || VALID || Keep | + | | mdh.TER.AP04 in pSB1CRDP #2 || #TVND# || #OX44# || 19 || VALID || Keep |
| |- | | |- |
− | | MDH.TER.AP04 in pSB1CRDP #3 || #1S1V# || #P9HA# || 20 || VALID || Discard | + | | 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 #1 || #HBWZ# || #BY9L# || 21 || VALID || Discard |
| |- | | |- |
− | | MDH.TER.AP19 in pSB1CRDP #2 || #K3V6# || #DNRY# || 22 || VALID || Discard | + | | mdh.TER.AP19 in pSB1CRDP #2 || #K3V6# || #DNRY# || 22 || VALID || Discard |
| |- | | |- |
− | | MDH.TER.AP19 in pSB1CRDP #3 || #CYF6# || #KV18# || 23 || VALID || Keep | + | | 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 #3 || #M4H8# || #ERZK# || 24 || VALID || Discard |
| |- | | |- |
− | | HPS.B1006.AP00 in pSB1CRDP #4 || #44WP# || #WXO8# || 25 || VALID || Keep | + | | 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.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 #2 || #RSH9# || #DERR# || 27 || MUTATION || Discard |
| |- | | |- |
− | | HPS.B1006.AP04 in pSB1CRDP #3 || #1C8L# || #DVRP# || 28 || 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.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 #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 #5 || #FB9L# || #39CF# || 43 || MUTATION || Discard
| + | |
− | |-
| + | |
− | | PHI.B1002.AP19 in pSB1CRDP #6 || #HWM4# || #RABS# || 44 || VALID || Keep
| + | |
− | |}
| + | |
− | | + | |
− | ===Noon===
| + | |
− | Prep plasmids of T7.POLY in BL21 (DE3)
| + | |
− | | + | |
− | {|class="wikitable"
| + | |
− | ! construct & clone number !! cryo ID !! plasmid ID !! check PCR result
| + | |
− | |-
| + | |
− | | T7.lacO.POLY in pSB1KRDP #1 || #LHBN# || #VYCO# || discard
| + | |
− | |-
| + | |
− | | T7.lacO.POLY in pSB1KRDP #5 || #HB93# || #ANPV# || discard
| + | |
− | |-
| + | |
− | | T7.lacO.POLY in pSB1KRDP #6 || #6LOZ# || #96TS# || discard
| + | |
− | |-
| + | |
− | | T7.lacO.POLY in pSB1KRDP #7 || #KYOQ# || #XVE3# || discard
| + | |
− | |-
| + | |
− | | T7.lacO.POLY in pSB1KRDP #8 || #MCMR# || #BSLA# || discard
| + | |
− | |}
| + | |
− | | + | |
− | * 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
| + | |
− | |-
| + | |
− | | || 2 || #HSP1# + #XSP3#|| 1939 || negative
| + | |
− | |-
| + | |
− | | || 3 || #XSP2# + #94LB#|| 2182 || negative
| + | |
− | |-
| + | |
− | | #ANPV# || 4 || #W6WP# + #HSP2#||1927 || negative
| + | |
− | |-
| + | |
− | | || 5 || #HSP1# + #XSP3#||1939 || negative
| + | |
− | |-
| + | |
− | | || 6 || #XSP2# + #94LB#||2182 || negative
| + | |
− | |-
| + | |
− | | #96TS# || 7 || #W6WP# + #HSP2#||1927 || negative
| + | |
− | |-
| + | |
− | | || 8 || #HSP1# + #XSP3#||1939 || negative
| + | |
− | |-
| + | |
− | | || 9 || #XSP2# + #94LB#||2182 || negative
| + | |
− | |-
| + | |
− | | #XVE3# || 10 || #W6WP# + #HSP2#||1927 || negative
| + | |
− | |-
| + | |
− | | || 11 || #HSP1# + #XSP3#||1939 || negative
| + | |
− | |-
| + | |
− | | || 12 || #XSP2# + #94LB#||2182 || negative
| + | |
− | |-
| + | |
− | | #BSLA# || 13 || #W6WP# + #HSP2#||1927 || negative
| + | |
− | |-
| + | |
− | | || 14 || #HSP1# + #XSP3#||1939 || negative
| + | |
− | |-
| + | |
− | | || 15 || #XSP2# + #94LB#||2182 || negative
| + | |
− | |-
| + | |
− | | #POLY# || 16 || #W6WP# + #HSP2#||1927 || positive
| + | |
− | |-
| + | |
− | | || 17 || #HSP1# + #XSP3#||1939 || positive
| + | |
− | |-
| + | |
− | | || 18 || #XSP2# + #94LB#||2182 || positive
| + | |
− | |}
| + | |
− | * Label yesterdays SDS gel and upload the results to the internal wiki.
| + | |
− | | + | |
− | ===Afternoon===
| + | |
− | * make M9+K+Glucose40mM overnight cultures of
| + | |
− | ** 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# || #....# | + | | phi.B1002.AP19 in pSB1CRDP #2 || #94OY# || #BND4# || 42 || NO CONTIGS FOUND || Discard |
| |- | | |- |
− | | AP19.MDH.Ter in pSB1KRDP || #WDXZ#, #FDX4#, #MOMX# || #....# | + | | phi.B1002.AP19 in pSB1CRDP #5 || #FB9L# || #39CF# || 43 || MUTATION || Discard |
| |- | | |- |
− | | pSB1KRDP || #QWRC# || #....# | + | | phi.B1002.AP19 in pSB1CRDP #6 || #HWM4# || #RABS# || 44 || VALID || Keep |
| |} | | |} |
| | | |