Melbourne/Lab Notebook
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====Transformation==== | ====Transformation==== | ||
#resuspended the following from Registry plates: | #resuspended the following from Registry plates: | ||
| - | #* | + | #*'''P2 21A''' - (BBa_I15008, ho1, Kan) |
| - | #*BBa_I15009 - PcyA (P2 21C, Kan) | + | #*BBa_I15009 - PcyA ('''P2 21C''', Kan) |
| - | #*BBa_R0084 - OmpR positive promoter (P1 11H, Amp) | + | #*BBa_R0084 - OmpR positive promoter ('''P1 11H''', Amp) |
#Punctured foil with pipette tip. | #Punctured foil with pipette tip. | ||
#Resuspended in 15uL ddH2O. | #Resuspended in 15uL ddH2O. | ||
| Line 27: | Line 27: | ||
====Transformation==== | ====Transformation==== | ||
#Resuspended the following parts in 15uL: | #Resuspended the following parts in 15uL: | ||
| - | ##BBa_B0034 (RBS, | + | ##BBa_B0034 (RBS, '''P1 3O''', Amp) |
| - | ##BBa_C0051 (C1 repressor, | + | ##BBa_C0051 (C1 repressor, '''P1 5G''', Amp) |
| - | ##BBa_B0010 (Terminator, | + | ##BBa_B0010 (Terminator, '''P2 3P''', Amp) |
*Think some DNA may have remained in wells | *Think some DNA may have remained in wells | ||
#[[Melb:Transformation Protocol|Transformed]] into competent DH5alpha cells from Joe | #[[Melb:Transformation Protocol|Transformed]] into competent DH5alpha cells from Joe | ||
Revision as of 06:03, 3 July 2007
Contents |
Week 1
25 June 2007
Prepared LB agar plates.
Transformation
- resuspended the following from Registry plates:
- P2 21A - (BBa_I15008, ho1, Kan)
- BBa_I15009 - PcyA (P2 21C, Kan)
- BBa_R0084 - OmpR positive promoter (P1 11H, Amp)
- Punctured foil with pipette tip.
- Resuspended in 15uL ddH2O.
- Stored in-20 (after taking 1uL for transformation).
- Transformed into competent DH5alpha cells with shorter incubation times as follows:
- 30min on ice after DNA addition
- 10min on ice after heat shock
- 30min at 37degrees with LB
26 June 2007
Transformation from Monday
- Transformation of BBa_I15008 and BBa_I15009 failed. No colonies on plates
- Small number of colonies on BBa_R0084 plate.
- Placed in cool room
Transformation
- Resuspended the following parts in 15uL:
- BBa_B0034 (RBS, P1 3O, Amp)
- BBa_C0051 (C1 repressor, P1 5G, Amp)
- BBa_B0010 (Terminator, P2 3P, Amp)
- Think some DNA may have remained in wells
- Transformed into competent DH5alpha cells from Joe
Streak plates
- Streaked the following cells:
- PJS010 (from solid agar, Amp)
- Fusion protein (from glycerol stock, Amp?)
- BBa_I15010 (from solid agar, Kan)
27 June 2007
Transformation
- Repeated transformation of failed parts from Monday:
- BBa_I15008 (Kan)
- BBa_I15009 (Kan)
- Used resuspended DNA that was stored on Monday
Liquid culture
- Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
- Aliquoted 5mL Amp LB into 6 50mL falcon tubes
- To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
- PJS010
- Fusion
- BBa_B0034
- BBa_C0051
- BBa_B0010
- BBa_R0084
- To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
- Cells incubated at 37degrees with shaking overnight.
28 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- PJS010
- Fusion
- BBa_B0034
- BBa_C0051
- BBa_B0010
- BBa_R0084
- BBa_I15010
- Stored in -20 freezer
Digest
Liquid culture
- Cultured the following cells from transformed plates:
- BBa_R0084
- BBa_I15010
- BBa_B0034
- BBa_C0051
- BBa_I15008
- BBa_I15009
29 June 2007
Miniprep
- Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
- BBa_R0084
- BBa_I15010
- BBa_B0034
- BBa_C0051
- BBa_I15008
- BBa_I15009
- Stored in -20 freezer
