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Team:HKU HongKong/Data/Weekly Notebook.html
From 2012.igem.org
Team:HKU HK
From 2011.igem.org
Daily Lab Notebookd
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Weeks 1&2:
1)
Amplification of pvdQ gene using PCR from Pseudomonas aeuriginosa genomic DNA. The designed primers contain the prefix and suffix so that typical digestion for insertion into iGEM’s standard vector can be subsequently performed.
2)Test digestion of amplified pvdQ with EcoRI only, EcoRI & SpeI, PstI, and EcoRI & XbaI.
3) Repeat the digestion of pvdQ with EcoRI and SpeI multiple times to obtain the desirable conditions of the digestion to prevent Star Activity.
4)Mass single digestion of the amplified pvdQ with EcoRI. Desirable fragment size should be about 5,600bp. The fragments that result due to Star activity are 3,800 and 1,800bp.
5)Gel purification and Nano-drop concentration check of digested pvdQ.
Weeks 3:
28/07/2012
1) Transform J23119 (constitutive promoter) and B0034 (Ribosomal Binding Site) from the kit.
2) Culture colonies from the transformation of ligated pvdQ-B0015.
28/07/2012
1) Transform J23119 (constitutive promoter) and B0034 (Ribosomal Binding Site) from the kit.
2) Culture colonies from the transformation of ligated pvdQ-B0015.
28/07/2012
1) Culture colonies from J23119 and B0034 transformation.
2) Colony PCR to confirm successful ligation of pvdQ-B0015.
3) Test digestion with enzymes
4) Gel electrophoresis of the digested volume of DNA (about 1ug)
to check correct band size.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
31/07/2012
1) Redo digestion of the J23119 plasmid using a greater amount of starting DNA (20uL) in a 50uL digestion reaction.
2) Perform electrophoresis and correct out the now-visible fragment of correct size and acceptable intensity.
3) Gel purify the J23119 fragment.
02/08/2012
1) Use nanodrop to check the concentration of previously digested B0034 as well as J23119.
2) Set up a suitable digestion reaction with J23119 as the insert and B0034 as the vector. Perform the ligation and incubate at room temperature for one hour.
3) Transform the ligated product.
03/08/2012
1) Culture the visible colonies on the transformed plate. Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked.
Result: The transformed plate had minisatellite colonies. Hence very few colonies could be picked.
2) Perform colony PCR.
Result: Expected amplified product’s size is similar to the band produced from primer dimerization. Hence the presence of the amplified product cannot be confirmed.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
30/07/2012
1) Miniprep the confirmed culture to extract J23119 and B0034 plasmid.
2) Restriction digestion with EcoRI and SpeI of J23119. Desired fragment size is 32 bp.
3) Restriction digestion of B0034 with EcoRI and XbaI. Desired fragment size is about 2,060bp.
4) Gel electrophoresis to cut out correct fragment size.
Result: While the B0034 fragment was large enough to be cut out and had a desirable intensity, the relatively small J23119bp fragment was not visible. This is because the majority of the DNA used in the digested product makes up the remaining 2,049bp backbone fragment.
