Team:BYU Provo/Notebook/Biofilm/mayjune

From 2014.igem.org

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<h2>1 May 2014</h2>
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<p>Aiia: Today a plasmid prep of the Aiia plasmid was performed following the Denville SpinSmart Plasmid Purification protocol.</p>
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<p>Alpha Amylase: Today sewing PCR was performed on the alpha amylase forward primer pieces. In order to PCR with the overlap extension primers that contain our signal sequence we will need to PCR with just the forward and reverse with the signal sequence and no template so that we have a combined forward primer to use. Then we will use that combined primer as a forward primer in a normal Q5 PCR reaction with the template. The only difference is that 2 uL of each primer should be used (the forward signaling sequence primer and the reverse signaling sequence primer). (JB)</p>
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<h2>2 May 2014</h2>
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<p>Today we ran our sewing PCR products on agarose gel and each got a product around 150 base pairs as it should have if the reaction worked properly. We then set up the next reaction with the newly sewn forward primers with the signaling sequence and the reverse primers for each gene as well as the template. We have a 5 uL control for each and then a reaction for each of the parts (including the two Amylase parts we are testing). The tube labeled Amy1 is pIG 92 and the tube labeled Amy2 is pIG98.</p>
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<h2>5 May 2014</h2>
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<p>Today we ran the gel of our PCR products of the enzyme plasmids with our sewn forward and regular reverse primers.</p>
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<p>Aiia had a product that was around 2500 bp, where is should have only been around 800 so we need to figure out if it amplified the whole plasmid.</p>
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<p>DispB showed smearing so either we need to play with the annealing temperatures because the primers are either not attaching at this temperature we used or the plasmid needs to be purified more.</p>
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<p>Amy2 was the best band. This was from PIG98. We will need to digest this and the backbone and run it on low melt gel.</p>
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<p>Left: Gel image of the sewing PCR reaction </p>
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<p>Right: Gel image of finished gene+signaling sequence PCR products (Lane order: 5 kb ladder, Aiia, Amylase1/pIG92, DispersinB, Amylase2/pIG98)</p>
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<h2>6 May 2014</h2>
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<p>Alpha Amylase: Today I purified the PCR product from the Alpha Amylase pIG98 plasmid (Amylase2) since it appeared to have the more solid band of the two on the gel. I then prepped a vector and insert digest from the purified PCR product and the iGem backbone. Thursday I will run it out on a low melt gel in order to perform a ligation of the two together. (JB)</p>
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<h2>8 May 2014</h2>
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<p>Alpha Amylase: Today I ran the low melt gel of the psb1c3 backbone and AA insert. It turned out well and I was able to see that a segment corresponding to the length of the RFP gene was separated from the plasmid. I followed our standard ligation procedure and will allow it sit overnight in order to get a better ligation. Tomorrow I will transform it into E. coli. (JB)</p>
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Revision as of 03:52, 24 July 2014


BYU 2014 Notebook

Edit May June

Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

1 May 2014

Aiia: Today a plasmid prep of the Aiia plasmid was performed following the Denville SpinSmart Plasmid Purification protocol.

Alpha Amylase: Today sewing PCR was performed on the alpha amylase forward primer pieces. In order to PCR with the overlap extension primers that contain our signal sequence we will need to PCR with just the forward and reverse with the signal sequence and no template so that we have a combined forward primer to use. Then we will use that combined primer as a forward primer in a normal Q5 PCR reaction with the template. The only difference is that 2 uL of each primer should be used (the forward signaling sequence primer and the reverse signaling sequence primer). (JB)

2 May 2014

Today we ran our sewing PCR products on agarose gel and each got a product around 150 base pairs as it should have if the reaction worked properly. We then set up the next reaction with the newly sewn forward primers with the signaling sequence and the reverse primers for each gene as well as the template. We have a 5 uL control for each and then a reaction for each of the parts (including the two Amylase parts we are testing). The tube labeled Amy1 is pIG 92 and the tube labeled Amy2 is pIG98.

5 May 2014

Today we ran the gel of our PCR products of the enzyme plasmids with our sewn forward and regular reverse primers.

Aiia had a product that was around 2500 bp, where is should have only been around 800 so we need to figure out if it amplified the whole plasmid.

DispB showed smearing so either we need to play with the annealing temperatures because the primers are either not attaching at this temperature we used or the plasmid needs to be purified more.

Amy2 was the best band. This was from PIG98. We will need to digest this and the backbone and run it on low melt gel.

Left: Gel image of the sewing PCR reaction

Right: Gel image of finished gene+signaling sequence PCR products (Lane order: 5 kb ladder, Aiia, Amylase1/pIG92, DispersinB, Amylase2/pIG98)

6 May 2014

Alpha Amylase: Today I purified the PCR product from the Alpha Amylase pIG98 plasmid (Amylase2) since it appeared to have the more solid band of the two on the gel. I then prepped a vector and insert digest from the purified PCR product and the iGem backbone. Thursday I will run it out on a low melt gel in order to perform a ligation of the two together. (JB)

8 May 2014

Alpha Amylase: Today I ran the low melt gel of the psb1c3 backbone and AA insert. It turned out well and I was able to see that a segment corresponding to the length of the RFP gene was separated from the plasmid. I followed our standard ligation procedure and will allow it sit overnight in order to get a better ligation. Tomorrow I will transform it into E. coli. (JB)





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