User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/28: Difference between revisions
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==Objective== | ==Objective== | ||
Troubleshoot ligation (see [[../24#Conclusions| notes from last week]] | Troubleshoot ligation (see [[../24#Conclusions| notes from last week]]) | ||
* transform old ligation into commercial cells | * transform old ligation into commercial cells | ||
* re-ligate O/N @ 16°C | * re-ligate O/N @ 16°C | ||
* start double-digest again | |||
Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready) | Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready) | ||
==Bench work== | ==Bench work== | ||
# | # Double-digest | ||
{| border="1" | |||
= | |- | ||
* | !Tube !!sterile H<sub>2</sub>O !!10X NEB2 !!100X BSA !!DNA !!NheI (10 kU/mL) !!SapI (2 kU/mL | ||
|- | |||
!pTXB1His | |||
|34.5 μL ||5 μL ||0.5 μL ||10 μL of 102 μg/mL ||2.5μL ||2.5μL | |||
|- | |||
!pTXB1 | |||
|34.5 μL ||5 μL ||0.5 μL ||10 μL of 82 μg/mL ||2.5μL ||2.5μL | |||
|- | |||
!BSA PCR | |||
|24.5 μL ||5 μL ||0.5 μL ||15 μL from purified PCR reaction ||2.5μL ||2.5μL | |||
|} | |||
:*→ 2h @ 37°C | |||
:*→ 30' @ 65°C | |||
:** 20' is sufficient | |||
:*→ store insert reaction @ -20°C | |||
#<li value="2">Phosphatase vectors | |||
#* add 1μL Alkaline Phosphatase to double-digest of pTXB1His and pTXB1 from step #1 | |||
#*→ 15' @ 37°C | |||
#*→ store @ -20°C | |||
# Transformation into [[NovaBlue]] | |||
#* Re-transform ligation from [[../22| last week]] into [[User:Matt Hartings|Matt Hartings]]' commercially competent NovaBlue ''E. coli'' cells | |||
## 5μL pTXB1His+BSA ligation + 50μL cells | |||
## 5μL pTXB1+BSA ligation + 50μL cells | |||
#* Followed standard transformation protocol for each sample in step #1 | |||
#*→ heat shock step was 45" | |||
#*→ plated 100μL each on LBAmp and stored the rest @ 4°C | |||
#**→ 37°C O/N | |||
*'''[[User:Kathryn Muratore|Kathryn Muratore]] 15:49, 29 June 2011 (EDT)''': I forgot to do the O/N ligation @ 16°C! | |||
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Revision as of 12:49, 29 June 2011
 AU CHEM-570 Lab Prep
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ObjectiveTroubleshoot ligation (see notes from last week)
Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready) Bench work
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