Team:Heidelberg/AwesomeSheet/Dnmt1 Methylation Assay

From 2014.igem.org

– Dnmt1 Methylation Assay

Awesome Sheet PA Gels Dnmt1 Methylation Assay Blots Waste SURPREMErun Strains Stocks Samples Purifications Plate Reader Marker LIGHTRun Enzymes Competent cells Induction Growth 3AL AGels AssmProd Assembly GoldenGate Parts Submission Parts PCR products PCRs Plasmids Plates Primer Res SL Trafos

ABCDEFGHIJKLMNOPQRSTUVWXYZAAABACADAEAFAGAHAIAJAKALAMANAOAPAQ
1
Gel LaneDate
Experimentator
purpose of experiment
Enzyme
Buffer
Template
SAMH2OTotal
Pre-incubation
Renaturing
Methylation
Methylation Stop
Restriction
GelResultCommentsImage
2
IDIDLabelDefBatchV/µlDefV/µlDefV/µlc/MV/µlc/MV/µlV/µlT/°Ct/sT/°Ct/sT/°Ct/sT/°Ct/sEnzymeV/µlc/Mt/sBufferV/µltotalTypeSDSVolt/Vt/minpngtiff
3
1M8/9/2014
first test of template + restrictione enzymes without Dnmt1
noneTAE 20%none8060blurry bands
4
18/9/2014allme0.2none0.210TAE 20%none8060
5
18/9/20141CRFme (semi)0.2none0.210TAE 20%none8060
6
18/9/20141CRme0.2none0.210TAE 20%none8060
7
18/9/2014unme0.2none0.210TAE 20%none8060
8
18/9/2014allme0.2Sau3AI0.21h1.1110TAE 20%none8060
9
18/9/20141CRFme (semi)0.2Sau3AI0.21h1.1110TAE 20%none8060
10
18/9/20141CRme0.2Sau3AI0.21h1.1110TAE 20%none8060
11
18/9/2014unme0.2Sau3AI0.21h1.1110TAE 20%none8060
12
18/9/2014allme0.2HpaIII0.215 minCutSmart110TAE 20%none8060
13
18/9/20141CRFme (semi)0.2HpaIII0.215 minCutSmart110TAE 20%none8060
14
18/9/20141CRme0.2HpaIII0.215 minCutSmart110TAE 20%none8060
15
18/9/2014unme0.2HpaIII0.215 minCutSmart110TAE 20%none8060
16
2M8/10/2014TAE 20%even more blurry bands
17
28/10/2014allme0.4none0.210TAE 20%none8090
18
28/10/20141CRFme (semi)0.4none0.210TAE 20%none8090
19
28/10/20141CRme0.4none0.210TAE 20%none8090
20
28/10/2014unme0.4none0.210TAE 20%none8090
21
28/10/2014allme0.4Sau3AI0.21h1.1110TAE 20%none8090
22
28/10/20141CRFme (semi)0.4Sau3AI0.21h1.1110TAE 20%none8090
23
28/10/20141CRme0.4Sau3AI0.21h1.1110TAE 20%none8090
24
28/10/2014unme0.4Sau3AI0.21h1.1110TAE 20%none8090
25
28/10/2014allme0.4HpaIII0.215 minCutSmart110TAE 20%none8090
26
28/10/20141CRFme (semi)0.4HpaIII0.215 minCutSmart110TAE 20%none8090
27
28/10/20141CRme0.4HpaIII0.215 minCutSmart110TAE 20%none8090
28
28/10/2014unme0.4HpaIII0.215 minCutSmart110TAE 20%none8090
29
38/12/2014
change in Polyacrylamid gel type to reduce blurryness of gels
allme0.4
30
38/12/20141CRFme (semi)0.4noneTBE 20%none70V60nice sharp bands, restriction enzymes do not cut DNA template completly and something like a oligo dimer was visible.
31
38/12/20141CRme0.4noneTBE 20%none70V60
32
38/12/2014unme0.4noneTBE 20%none70V60
33
38/12/2014allme0.4noneTBE 20%none70V60
34
38/12/20141CRFme (semi)0.4Sau3AI0.32h1.1110TBE 20%none70V60
35
38/12/20141CRme0.4Sau3AI0.32h1.1110TBE 20%none70V60
36
38/12/2014unme0.4Sau3AI0.32h1.1110TBE 20%none70V60
37
38/12/2014allme0.4Sau3AI0.32h1.1110TBE 20%none70V60
38
38/12/20141CRFme (semi)0.4HpaIII0.32h1.1110TBE 20%none70V60
39
38/12/20141CRme0.4HpaIII0.32h1.1110TBE 20%none70V60
40
38/12/2014unme0.4HpaIII0.32h1.1110TBE 20%none70V60
41
3M8/12/2014HpaIII0.32h1.1110TBE 20%none70V60
42
4first activity assay of linear Dnmt1, procedure as seen in “Specificity of Dnmt1 for Methylation of Hemimethylated CpG Sites Resides in Its Catalytic Domain” by P. Bashtrykov published in Chemistry & Biology 19, 572–578, May 25, 2012 http://www.ncbi.nlm.nih.gov/pubmed/22633409 ctrlunme37
overnight
43
4ctrl1CRme37
overnight
44
4ctrl1CRFme (semi)37
overnight
45
4Dnmt11AHEPES1CRme37
overnight
46
4Dnmt11BHEPES1CRme37
overnight
47
4Dnmt11CHEPES1CRme37
overnight
48
4Dnmt12HEPES1CRme37
overnight
49
4Dnmt12AHEPES1CRme
50
4Dnmt13HEPES1CRme37
overnight
51
4Dnmt14HEPES1CRme
52
4M
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5M
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Dnmt14
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Dnmt13
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Dnmt1
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Dnmt1
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Dnmt1
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Dnmt1
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Dnmt1
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Retrieved from "http://2014.igem.org/Team:Heidelberg/AwesomeSheet/Dnmt1_Methylation_Assay"