User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/16: Difference between revisions
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==Objective== | ==Objective== | ||
* Run QuikChange on pMXB10 from [[../15#Bench work|yesterday]](by Dr. Muratore) on gel | |||
* Check transformations on plates | |||
* Prepare for expression of pMAL-pIII and pMXB10/start cultures from colonies on plates | |||
==Bench work== | ==Bench work== | ||
# | # Ran 1.0% agarose DNA gel with [[Muratore:Protocols/PCR/QuikChange | 2-stage QuikChange]] of pMXB10 for +His tag from [[../15#Bench work|yesterday]](by Dr. Muratore) | ||
#* Lane 1: Ladder | |||
#* Lane 2: ---- | |||
#* Lane 3: ---- | |||
#* Lane 4: pMXB10 QC +His [[../15 | 8/15]] | |||
#* Lane 5: ---- | |||
#* Lane 6: ---- | |||
#* Lane 7: pMXB10 QC Ctrl [[../15 | 8/15]] | |||
# Due to perceived failure of QuikChange from yesterday, performed a test PCR with pCMV-SPORT6+BSA to amplify BSA gene | |||
#* Vary Pfu Turbo buffer and Pfu Turbo polymerase in PCR reaction: | |||
#* Buffer A + Polymerase 1 == 'A1' | |||
#* Buffer A + Polymerase 2 == 'A2' | |||
#* Buffer B + Polymerase 1 == 'B1' | |||
#* Buffer B + Polymerase 2 == 'B2' | |||
#* Follow same protocol as [[../../05/26 | 5/26]] | |||
# Took 1 colony from each of pMAL-pIII and pMXB10 in DH10B and put into 10mL of LB medium with 10μL of 100mg/mL ampicillin | |||
#* Grew O/N at 37°C | |||
# Took 1 colony from each of pMAL-PIII and pMXB10 in ER2566 and put into 50mL of LB medium with 50μL of 100mg/mL ampicillin | |||
#* Grew O/N at 37°C | |||
# Replated the four lowest-yielding transformations: | |||
#* pMAL-pIII and pMXB10 in ER2566 | |||
#* BamHI-digested pKK223-3 ligations of 10μL and 15μL in DH10B | |||
==Results== | ==Results== | ||
==Gel== | |||
[[Image: DNA_gel_pMXB10_8.16.2011_labeled.jpg]] | |||
**Did not see what we wanted on gel -- suspect issue with reagents | |||
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Revision as of 11:26, 17 August 2011
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Objective
Bench work
ResultsGel
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