SBB10Ntbk-JennaKelleher: Difference between revisions
No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
[[SBB10Ntbk- | ==2/22/2010== | ||
[[ | ===Labwork=== | ||
Last time: basic PCR for sbb28, sbb29 <br> | |||
This time: ran 2 analytical gels <br> | |||
3 microliters PCR | |||
7 microliters Loading Buffer | |||
While waiting, ran zymo cleanup on the 2 samples, final volume of 30.6 microliters<br> | |||
Stored in 140L '''Box A''' <br> | |||
<br> | |||
Next time: digestion | |||
===Gel Results=== | |||
Analytical gel images shown below (ladder breakdown on left, gels on right), loaded and imaged by [[SBB10Ntbk-DorothyTulanont| Dorothy Tulanont]]<br> | |||
<br> | |||
[[Image:Generuler1kbplus.jpg]] | |||
[[Image:DDT_02_22_10.jpg]]<br> | |||
<br> | |||
Relevant Lanes: | |||
#ladder | |||
#sbb28 - looks to be about 1500 bp | |||
#sbb29 - looks to be about 1000 bp | |||
Both of these values are close to desired lengths, so PCR worked!<br> | |||
<br> | |||
==2/17/2010== | |||
===Labwork=== | ===Labwork=== | ||
| Line 36: | Line 57: | ||
Product is pBjk2741-Bjh1723 {rbs_pelB>} | Product is pBjk2741-Bjh1723 {rbs_pelB>} | ||
==2/10/2010== | |||
===Project Information=== | ===Project Information=== | ||
[[Template:SBB10Project-29071 | 29071-O99 rep Protein, CA42 rep Gene, Pre-Pro sequence]]<br> | [[Template:SBB10Project-29071 | 29071-O99 rep Protein, CA42 rep Gene, Pre-Pro sequence]]<br> | ||
Revision as of 12:46, 24 February 2010
2/22/2010
Labwork
Last time: basic PCR for sbb28, sbb29
This time: ran 2 analytical gels
3 microliters PCR 7 microliters Loading Buffer
While waiting, ran zymo cleanup on the 2 samples, final volume of 30.6 microliters
Stored in 140L Box A
Next time: digestion
Gel Results
Analytical gel images shown below (ladder breakdown on left, gels on right), loaded and imaged by Dorothy Tulanont
Relevant Lanes:
- ladder
- sbb28 - looks to be about 1500 bp
- sbb29 - looks to be about 1000 bp
Both of these values are close to desired lengths, so PCR worked!
2/17/2010
Labwork
Prepared sbb28 and sbb29 for PCR after creating a master mix with Jeni Lee
sbb28 in 2k55 and sbb29 in 55
Updated Constuction Files
Biobricking of 099 rep protein basic part sbb29
PCR repo99-F and JG002-R on pEC52 gene (978 bp, EcoRI/BamHI)
Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L)
Product is pBca9523-sbb29 {rbs.repO99!}
----------------------------
repo99-F Cloning of repo99
ccaaaGAATTCatgAGATCTcctggaaggaatcgtaacac
JG002-R Cloning of O99 rep Gene
CTGATGGATCCCTACTCTACAAGACCTCGTTTTttc
Biobricking of CA42 rep gene basic part sbb28
PCR repCA42-F and KRM002 on pEC22-CA42 gene (1496 bp, EcoRI/BamHI)
Sub into pBca9523-Bca1144 (EcoRI/BamHI, 910+2472, L)
Product is pBca9523-sbb28 {5'UTR.repCA42!}
----------------------------
repCA42-F Cloning of repCA42
ccaaaGAATTCatgAGATCTcagctgaagtgaccggattag
KRM002 Reverse oligo for cloning of rbs.repCA42!
ctgatGGATCCctattttccgcttttccag
Biobricking of pre-pro sequence basic part sbb42
Eco/Bam transfer pBjh1601AC-Bjh1723
Sub into pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-Bjh1723 {rbs_pelB>}
2/10/2010
Project Information
29071-O99 rep Protein, CA42 rep Gene, Pre-Pro sequence
Construction Files
Jenna Kelleher 099 Rep Protein (sbb29)
Jenna Kelleher CA42 Rep Gene (sbb28)
Jenna Kelleher Pre-pro Sequence (sbb42)
Useful Protocols
For SBB28 and SBB29:
Cloning by PCR
Regular Zymo Cleanup
For SBB42:
Eco-Bam Transfer
Small-Frag Zymo Cleanup
For All 3:
EcoRI/BamHI Digest of PCR Products
Zymo Gel Purification
Ligation of EcoRI/BamHI digests
Transformation by heat-shock
Picking of colonies
Macherey-Nagel Miniprep
EcoRI/BamHI Digest of PCR Products
Analytical digests (Mapping)
