Difference between revisions of "Team:SPSingapore/Notebook-Week-6"

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&#9642; July 4
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<span class = "brown"> Anaerobic Promoter</span>
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&emsp;&emsp;
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<font style = "color:blue;font-weight:bold;font-size:11pt;">&#9728; Yi Han &#9728;</font>
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No colonies-> ligation did not work
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Recalculate ligation reaction (3:1),7x
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<tr><td> 350ng vector->9ul vector </td></tr>
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<tr><td> 446.3ng insert-> 2ul </td></tr>
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<tr><td> 110 H2O </td></tr>
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<tr><td> 12ul buffer </td></tr>
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&#9654; ligate for 2 hours at 16 hours
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<br> &#9654; transform, plate, grow ON
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Revision as of 20:34, 17 September 2015


Research Notebook

Week 6 (28/6 - 4/7)

▪ June 28

esa Quorum Sensing    ☀ Adrian ☀

BBPrefix_esaRBS PCR
H2O 221ul
Buffer 80ul
MgCl1 32ul
dNTP 32ul
FP_Bioricks Prefix 16ul
ORP_esaRBS_fragsyn 16ul
synpart_BBP_esaRBS 1ul
gotaq 2ul
Total 400ul
▶ Success-> PCR purification

esaRBS_GFP_BBsuffix
H2O 237ul
Buffer 80ul
MgCl2 32ul
dNTP 32ul
RP_BB_suffix 16ul
ORP_esaRS_GFP 16ul
plac_GFPPLASMID 1ul
gotaq 2ul
▶ Failed -> troubleshooting-> new OFP_esaRBBS_GFP
    Redid 30/6 with new primers
    Success with new primers-> fusion pcr


▪ June 29

Lab Safety    ☀ SPSingapore Team ☀

Safety Inspection for our lab by OSHE. We passed with flying colours!


▪ July 2

esa Quorum Sensing    ☀ Clarice ☀

Fusion PCR with esaRBS, GFP
H2O 104.07ul
Buffer 40ul
MgCl2 16ul
dNTPs 16ul
FP_BB_prefix 8ul
RP_BB_prefix 8ul
400ng esaRBS 2.13ul
400ng esaRBSgfp 4.8ul
gotaq 1ul
total 200ul

▶ annealing temp 50degC
▶ Gel electrophoresis: looks correct-> PCR Purification

RE digest of esaRBS+GFP insert
Buffer 2
EcoRI/PstI 1/1
Template 2ug 5ul
dH2O 11ul
Total 20ul


RE digest of esaRBS+GFP vector
Buffer 2ul
EcoRI/PstI 1/1
Template 4ul 2ug 5ul
dH2O 12ul
Total 20ul


▪ July 3

Anaerobic Promoter    ☀ Yi Han ☀

Ran gel of gfp plasmid EcoRI/PstI, took out gel slice for vector - 4kb
▶ gel extraction
▶ gfpvector->38.7ng/ul
▶ fragment-> direction purification 226.4ng/ul

Ligation reaction (5:1) 6X
300ng vector 7.8ng/ul
531.2ng of insert 2.4ul
12ul buffer
6ul enz
91.8ul H2O

▶ ligate for 2 hours at 16 hours
▶ transform, plate, grow ON


▪ July 4

Anaerobic Promoter    ☀ Yi Han ☀

No colonies-> ligation did not work

Recalculate ligation reaction (3:1),7x
350ng vector->9ul vector
446.3ng insert-> 2ul
7ul ligase
110 H2O
12ul buffer

▶ ligate for 2 hours at 16 hours
▶ transform, plate, grow ON