User:Tk/Notebook/MF-xfm/2008/04/20: Difference between revisions
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(Autocreate 2008/04/20 Entry for User:Tk/Notebook/MF-xfm) |
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== | ==Transformation results== | ||
* | * 73 transformants from last week's transformations | ||
* Most from transformations with low cell density | |||
* Most from transformations with 10 ul 1M sorbitol added prior to electroporation | |||
* nothing shows at 14 hours or 24 hours | |||
* Some colonies visible at 38 hours, more at 48, final at 60 or so | |||
==New standard transformation protocol== | |||
* culture cells in 45 ml 1161 medium to light yellow/slightly turbid | |||
* Spin down and resuspend 3x with 45 ml EPB (no glycerol) on ice in chilled centrifuge | |||
* Resuspend in 5 ml EPB on ice | |||
* Aliquot 50 ul cells + 1 ul transposon DNA on ice | |||
* Add 10 ul 1 M sorbitol | |||
* Electroporate at 1.5 KV in 1 mm cuvette | |||
* Immediately add 850 ul 1161 medium, place on ice | |||
* Incubate 1 hour at 30C in the cuvette | |||
* Plate 300 ul on 15 ug/ml TET plates | |||
* Hold remainder in the cuvette for several days at 4C | |||
==Newly diluted transposase== | |||
* Dilute 50 ul transposase (8 ug/ul) into 800 ul transposase storage buffer | |||
* Store at -20, label 500 ng/ul transposase | |||
==New transposomes== | |||
* 20 ul transposon DNA (83 ng/ul) | |||
* 20 ul glycerol (use cut off tip for pipetting) | |||
* 40 ul diluted transposase | |||
* incubate 1 hour at 37C | |||
* hold at -20C | |||
==New transformations== | |||
* standard protocol | |||
** use 10 ul water, 5 ul water, no addition, 5 ul, 10 ul, and 15 ul sorbitol (1 M) additions | |||
* Test for both positive and negative osmotic pressure changes, and narrower range than last time | |||
* Plate out all of sorbitol added versions (3 plates) | |||
* Plate out only 1 plate from water and no addition plates, save remainder at 4C | |||
* Plates labeled with -10, -5, 0, +5, +10, +15 for water and sorbitol additions (ul added) | |||
==New plates and culture medium, standard protocol== | |||
==Plans for genetics== | |||
* Promoters | |||
** constitutive | |||
** heat shock | |||
* Reporters | |||
** YFP, mCherry | |||
* antibiotic | |||
** cat gene | |||
* recT recombination gene | |||
* Test constitutive and heat shock promoters with reporter | |||
* Test cat gene with constitutive promoter | |||
* Combine heat shock promoter with recT gene | |||
* Test recombination efficiency | |||
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Revision as of 21:52, 19 June 2008
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Transformation results
New standard transformation protocol
Newly diluted transposase
New transposomes
New transformations
New plates and culture medium, standard protocolPlans for genetics
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