Edinburgh/19 August 2008

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==== Tuesday 19 August ====
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==Week 10==
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=== Tuesday 19 August ===
*Results from yesterday's analytical digests of M144-155 (gel 55):  
*Results from yesterday's analytical digests of M144-155 (gel 55):  
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M144-147 (pSB1A2+cenA) all have a single band around 2.5 kb. Doesn't look promising, but perhaps this could have something to do with using methanol instead of ethanol for the miniprep? It seems unlikely as the undigested band was expected at ~3.5kb. I'll check this with Chris.  
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**M144-147 (pSB1A2+''cenA'') all have a single band around 2.5 kb. Doesn't look promising, but perhaps this could have something to do with using methanol instead of ethanol for the miniprep? It seems unlikely as the undigested band was expected at ~3.5kb. I'll check this with Chris.  
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M148-151 (pSB1A2+cex): M148, M149 and M151 all look promising. There is a band at about 3-3.5kb (undigested plasmid?), a band at ~2kb (digested plasmid) and a band around ~750bp (Remember that cex has a PstI site halfway through. M150 looks different, but also promising. There is a band at ~2kb and a smear a little smaller than 1kb. I suggest that M150 and M151 be submitted for sequencing asap. (AH)
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M148-151 (pSB1A2+''cex''): M148, M149 and M151 all look promising. There is a band at about 3-3.5kb (undigested plasmid?), a band at ~2kb (digested plasmid) and a band around ~750bp (Remember that cex has a PstI site halfway through. M150 looks different, but also promising. There is a band at ~2kb and a smear a little smaller than 1kb. I suggest that M150 and M151 be submitted for sequencing asap. (AH)
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**M152-155 (pSB1A2+''dxs''+''crtE''): M152 and M154 both have three bands at 5kb (undigested plasmid), 3kb (''dxs''+''crtE'') and 2kb (digested vector). M153 has a smaller largest band at ~4.5kb and a thickish band at about 2kb. M155's banding pattern is similar to M152 and M154's, but all the bands are ~500bp smaller. M152 should be submitted for sequencing. (AH)  
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M152-155 (pSB1A2+dxs+crtE): M152 and M154 both have three bands at 5kb (undigested plasmid), 3kb (dxs+crtE) and 2kb (digested vector). M153 has a smaller largest band at ~4.5kb and a thickish band at about 2kb. M155's banding pattern is similar to M152 and M154's, but all the bands are ~500bp smaller. M152 should be submitted for sequencing. (AH)  
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*Maxpreps from Plate 102(crtB/crtI) to X13, Plate 108(crtY-pSB1A2) to X14, Plate 108(rbs-crtY-pSB1A2) to X15, Plate115(PcstA) to X16 and Plate 115(dxs-lims) to X17. (HX)  
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*Maxipreps from Plate 102 (''crtB/crtI'') to X13, Plate 108 (''crtY''-pSB1A2) to X14, Plate 108 (rbs-''crtY''-pSB1A2) to X15, Plate115 (P''cstA'') to X16 and Plate 115(''dxs-lims'') to X17. (HX)  
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*Ligation of AppY into dxs+Lims (L48), and Appy into CrtBI (L49). The ligation of Lac Z into PcstA was not done, because Lac Z gene could not be purified! (YAN)  
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*Ligation of ''appY'' into ''dxs+LIMS'' (L48), and ''appY'' into ''crtBI'' (L49). The ligation of ''lacZ'' into P''cstA'' was not done because ''lacZ'' gene could not be purified! (YAN)  
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Made overnight culture of CenA (1-4) (YAN)  
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* Made overnight culture of ''cenA'' (1-4) (YAN)  
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*PCR of glgC+SOB2. P78~80: forward primer glgCf1, reverse primer SOBr1. P81~83: forward primer glgCf1, reverse primer SOB-rbs.  
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* PCR of ''glgC+SOB2''. P78~80: forward primer glgCf1, reverse primer SOBr1. P81~83: forward primer glgCf1, reverse primer SOB-rbs.
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P78: SOB2-glgC mut 1+2 from L45.  
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**P78: SOB2-''glgC'' mut 1+2 from L45.  
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P79: SOB2-glgC mut 1+2+3 from L46.  
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**P79: SOB2-''glgC'' mut 1+2+3 from L46.  
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P80: SOB2-glgC mut 1+2+3 from L47.  
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**P80: SOB2-''glgC'' mut 1+2+3 from L47.  
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P81: SOB2-rbs-glgc mut 1+2 from L45.  
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**P81: SOB2-rbs-''glgC'' mut 1+2 from L45.  
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P82: SOB2-rbs-glgC mut 1+2+3 from L46.  
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**P82: SOB2-rbs-''glgC'' mut 1+2+3 from L46.  
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P83: SOB2-rbs-glgC mut 1+2+3 from L47. (AM)
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**P83: SOB2-rbs-''glgC'' mut 1+2+3 from L47. (AM)
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'''[[Edinburgh/20_August_2008|Next Entry >]]'''
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Latest revision as of 21:43, 29 October 2008

< Previous Entry | Notebook Index | Next Entry >

Week 10

Tuesday 19 August

  • Results from yesterday's analytical digests of M144-155 (gel 55):
    • M144-147 (pSB1A2+cenA) all have a single band around 2.5 kb. Doesn't look promising, but perhaps this could have something to do with using methanol instead of ethanol for the miniprep? It seems unlikely as the undigested band was expected at ~3.5kb. I'll check this with Chris.

M148-151 (pSB1A2+cex): M148, M149 and M151 all look promising. There is a band at about 3-3.5kb (undigested plasmid?), a band at ~2kb (digested plasmid) and a band around ~750bp (Remember that cex has a PstI site halfway through. M150 looks different, but also promising. There is a band at ~2kb and a smear a little smaller than 1kb. I suggest that M150 and M151 be submitted for sequencing asap. (AH)

    • M152-155 (pSB1A2+dxs+crtE): M152 and M154 both have three bands at 5kb (undigested plasmid), 3kb (dxs+crtE) and 2kb (digested vector). M153 has a smaller largest band at ~4.5kb and a thickish band at about 2kb. M155's banding pattern is similar to M152 and M154's, but all the bands are ~500bp smaller. M152 should be submitted for sequencing. (AH)
  • Maxipreps from Plate 102 (crtB/crtI) to X13, Plate 108 (crtY-pSB1A2) to X14, Plate 108 (rbs-crtY-pSB1A2) to X15, Plate115 (PcstA) to X16 and Plate 115(dxs-lims) to X17. (HX)
  • Ligation of appY into dxs+LIMS (L48), and appY into crtBI (L49). The ligation of lacZ into PcstA was not done because lacZ gene could not be purified! (YAN)
  • Made overnight culture of cenA (1-4) (YAN)
  • PCR of glgC+SOB2. P78~80: forward primer glgCf1, reverse primer SOBr1. P81~83: forward primer glgCf1, reverse primer SOB-rbs.
    • P78: SOB2-glgC mut 1+2 from L45.
    • P79: SOB2-glgC mut 1+2+3 from L46.
    • P80: SOB2-glgC mut 1+2+3 from L47.
    • P81: SOB2-rbs-glgC mut 1+2 from L45.
    • P82: SOB2-rbs-glgC mut 1+2+3 from L46.
    • P83: SOB2-rbs-glgC mut 1+2+3 from L47. (AM)
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