Team:CityColSanFrancisco/Notebook/LabBooks/Bowen



     07172009





 07172009 

Goals:     PCR amplification of OprB from genomic DNA:  P. aeurginosa .

<p class=MsoNormal>                 <span class=GramE>Digestion of both plasmids.

<p class=MsoNormal>                 <span class=SpellE> Ligation of gene into plasmid for further alteration.

<p class=MsoNormal>                 <span class=GramE>Large liquid culture of <i style='mso-bidi-font-style:normal'>R. <span class=SpellE>palustris </i>.

<p class=MsoNormal>PCR reaction:</b> 10 reactions, 25&#956;L total volume each. Basic forward and reverse primers were used. Homegrown Taq polymerase was used. All tubes contained the same reaction. Exp003 of <span class=SpellE>thermocycler used.

<p class=MsoNormal>Plasmid Digestion:</b><span style='mso-spacerun:yes'>  4 reactions were done according to the plasmid digestion protocol. Enzymes used: EcoRI and SpeI.

<p class=MsoNormal>Gel Electrophoresis:</b> 1.0% gel was used to run both the PCR and Plasmid digestion.<span style='mso-spacerun:yes'>  Two separate gels were run.<span style='mso-spacerun:yes'>

<p class=MsoNormal style='margin-left:.5in'>PCR Gel:<span style='mso-spacerun:yes'>  9&#956;L of rxn w/ 2&#956;L loading dye. 10&#956;L was loaded into each of the lanes. 5&#956;l of 100bp ladder was also loaded.

<p class=MsoNormal style='margin-left:.5in'>Please refer to Figure 1 – not available.

<p class=MsoNormal style='margin-left:.5in'>Lane 1 – Ladder

<p class=MsoNormal style='margin-left:.5in'>Lane 2 – tube 2

<p class=MsoNormal style='margin-left:.5in'>Lane 3 – tube 4

<p class=MsoNormal style='margin-left:.5in'>Lane 4 - tube 6

<p class=MsoNormal style='margin-left:.5in'>Lane 5 – tube 8

<p class=MsoNormal style='margin-left:.5in'>Lane 6 – Ladder

<p class=MsoNormal>Results: No distinct banding was seen.<span style='mso-spacerun:yes'>  Although the ladder turned out quite well, no other valid banding is seen. Gel was included as it was the first that I had run.

<p class=MsoNormal style='margin-left:.5in'>Digestion Gel: 10&#956;L of <span class=SpellE>rxn w/ 2&#956;L of loading dye.<span style='mso-spacerun:yes'>  10&#956;L was loaded into was loaded into each of the lanes. 5&#956;L of 100bp ladder was loaded.

<p class=MsoNormal style='margin-left:.5in'>Please refer to figure 2.

<p class=MsoNormal style='margin-left:.5in'>Lane 1 – Ladder

<p class=MsoNormal style='margin-left:.5in'>Lane 2 – BBa_digest 1

<p class=MsoNormal style='margin-left:.5in'>Lane 3 – BBa_digest 2

<p class=MsoNormal style='margin-left:.5in'>Lane 4 – pSB_digest 1

<p class=MsoNormal style='margin-left:.5in'>Lane 5 – pSB_digest 2

<p class=MsoNormal style='margin-left:.5in'>Lane 6 – Ladder

<p class=MsoNormal>Results: Success!! BOTH plasmids were digested as planned. Thank you <span class=SpellE>iGEM for kits that work!! Although it seems that the banding is really close, the actual fragments of the digested plasmids are so similar that they would not be resolved on a 1.0% gel.<span style='mso-spacerun:yes'>  Perhaps on a 2.0% gel…

<p class=MsoNormal>Conclusion:   Because the PCR was a flop, it is necessary to look at the primers and see if there is a way to alter them. Cycling parameters are also looked at. Possibly annealing temp and time should be altered to better suit our primers.<span style='mso-spacerun:yes'>  After plenty of thought, it was discovered that one of the primers was designed with the biobrick extension sites added to the wrong side of the priming region.<span style='mso-spacerun:yes'>  Primers were re-ordered – in the correct order, with the same priming region.

<p class=MsoNormal> 07202009 </b>

<p class=MsoNormal>Cell Culture: </b>

<p class=MsoNormal>Liquid cultures of the <i style='mso-bidi-font-style:normal'>E. coli</i> containing plasmids were inoculated in liquid LB culture tubes w/ Morton closures. Two of six cultures were placed in a shaker incubator (37°C) and two were left out at room temp.

<p class=MsoNormal>Other cultures (all cell lines) were maintained.<span style='mso-spacerun:yes'>  Liquid cultures were made for frozen cell bank. Plates were created so they could be parafilmed and placed in a refrigerator for potential use once summer break is over. Old plates and tubes were disposed of via autoclave.

<p class=MsoNormal> 07212009 </b>

<p class=MsoNormal>Frozen stocks were created from overnight cultures.

<p class=MsoNormal> 07242009 </b>

<p class=MsoNormal>Planning day! Last day before summer vacation begins. Will resume work mid to late August. <span style='mso-spacerun:yes'>  Insert big frowny face here!

<p class=MsoNormal> 08262009 </b>

<p class=MsoNormal>Media Prep: LB, YP, and FEM media.<span style='mso-spacerun:yes'>  1L each. <span style='mso-spacerun:yes'>  Autoclaved and stored. <span style='mso-spacerun:yes'>  This media will be used to start large scale cultures for the battery chambers.

<p class=MsoNormal>                 *The vitamin solution and trace elements DO NOT get autoclaved!!!

<p class=MsoNormal>Cell Culture:   Liquid and plates were created of <i style='mso-bidi-font-style:normal'>R. <span class=SpellE>ferrireducins, Shewenella , </i>and <i style='mso-bidi-font-style:normal'>R. Palustris .</i>

<p class=MsoNormal> 09042009 </b>

<p class=MsoNormal>PCR for Point Mutagenesis: The forward primer w/ biobrick extensions is paired with the reverse mutagenesis primer (combo 1/6).<span style='mso-spacerun:yes'>  The reverse primer w/ biobrick extensions is paired with the forward mutagenesis primer (combo 5/7).<span style='mso-spacerun:yes'>  Exp003 was used in the thermocycler.

<p class=MsoNormal> 09092009 </b>

<p class=MsoNormal>   I was intending to run a gel, but for some reason, all of the PCR product had evaporated…I placed them in microfuge and pulsed them for quite some time. However, no product was recovered. I repeated the PCR protocol from 09042009. Hopefully, there will be product for running a gel on 09112009.

<p class=MsoNormal> <span style='text-decoration:none'> </b>

<p class=MsoNormal> 09112009 </b>

<p class=MsoNormal>Gel Electrophoresis:</b><span style='mso-spacerun:yes'>  1.0% agarose gel was used. 5&#956;L product was combined with an equal part of loading dye. All 10&#956;l were loaded onto the gel. 6&#956;L of 100bp ladder was also loaded.

<p class=MsoNormal>                 See Figure 3.

<p class=MsoNormal>                 Lane 1 - Blank

<p class=MsoNormal style='text-indent:.5in'>Lane 2 – Ladder

<p class=MsoNormal>                 Lane 3 – negative control

<p class=MsoNormal>                 Lane 4 – tube 1 – primer combo 5/7

<p class=MsoNormal>                 Lane 5 – tube 2 – primer combo 5/7

<p class=MsoNormal>                 Lane 6 – Blank

<p class=MsoNormal>                 Lane 7 - Ladder

<p class=MsoNormal>                 Lane 8 – tube 1 – primer combo 1/6

<p class=MsoNormal>                 Lane 9 – tube 2 – primer combo 1/6

<p class=MsoNormal>                 Lane 10 - Blank

<p class=MsoNormal>Results:   Although there is a LOT of extraneous banding, there IS a band in each of the lanes that is very promising! Lanes 4 and 5 have a bright lower band that corresponds to the size of the small product (<span class=SpellE>OprB gene w/ single base change) and the third band from the top of lanes 8 and 9 correspond to the larger product from point mutagenesis. This reaction needs to be optimized and then run on a prep gel for ban extraction and purification.<span style='mso-spacerun:yes'>  Then, the two pieces can be <span class=SpellE>ligated via PCR with only the primers containing the <span class=SpellE>biobrick extensions.

<p class=MsoNormal> 09162009 </b>

<p class=MsoNormal>Planning day!! We have attempted to set a schedule for the rest of the time that we have to work. If all goes well, this schedule will be a good one.<span style='mso-spacerun:yes'>  However, capturing the “gene in a jar” is harder than it seems! Our plasmids are digested and ready to go. The gene of interest is not.

<p class=MsoNormal> <span style='text-decoration:none'> </b>

<p class=MsoNormal> 09182009 </b>

<p class=MsoNormal>PCR:   Instead of optimizing the last reaction, it has been decided to combine the products of the last run and proceed with PCR in attempt to combine the fragments with the forward and reverse biobrick primers.<span style='mso-spacerun:yes'>  The ultimate goal is to capture the gene of interest with the restrictions sites added to the ends of the gene.<span style='mso-spacerun:yes'>  It will then get ligated into the plasmids and transformed into competent <i style='mso-bidi-font-style: normal'>E. coli </i>cells. Once the transformation has (hopefully) occurred, the plasmids will be harvested for transformation into <i style='mso-bidi-font-style:normal'>R. <span class=SpellE>palustris .</i> Exp004 on the thermocycler was used.<span style='mso-spacerun:yes'>  This raised the annealing temperature and extended the elongation time. Gels will be run next meeting.

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> 09212009 </b>

<p class=MsoNormal>Gel Electrophoresis: of 09182009 PCR reaction.

<p class=MsoNormal>                 See Figure 4.

<p class=MsoNormal>                 Lane 1 – Blank

<p class=MsoNormal>                 Lane 2 – Ladder

<p class=MsoNormal>                 Lane 3 – negative control

<p class=MsoNormal>                 Lane 4 – tube 1

<p class=MsoNormal>                 Lane 5 – tube 2

<p class=MsoNormal>                 Lane 6 – Blank

<p class=MsoNormal>Results:   Although faint bands are seen, there is no real product to work with.<span style='mso-spacerun:yes'>  In order to run a prep gel for band excision and purification, it is necessary to see a clear band of product.<span style='mso-spacerun:yes'>  Perhaps it is time to do a colony PCR.<span style='mso-spacerun:yes'>  This would use the genomic DNA from a colony directly. Perhaps there were errors in purifying the DNA…we shall see!

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> 09252009 </b>

<p class=MsoNormal>More PCR:   Colony PCR of <i style='mso-bidi-font-style:normal'>P. aeurignosa .</i><span style='mso-spacerun:yes'>  7 reactions, 25&#956;L each. See Excel spreadsheet for concentrations. MgCl2 added, regular PCR buffer used.

<p class=MsoNormal>Cycling info:

<p class=MsoNormal>95°C – 4 minutes

<p class=MsoNormal>92°C – 30 seconds

<p class=MsoNormal>50°C – 30 seconds

<p class=MsoNormal>72°C – 90 seconds

<p class=MsoNormal>72°C – 5 minutes

<p class=MsoNormal> 4°C – forever.

<p class=MsoNormal>29 cycles were done (temps 2, 3, and 4).<span style='mso-spacerun:yes'>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> 09282009 </b>

<p class=MsoNormal>Gel Electrophoresis: A 1.0% gel was run. It yielded nothing. Only the ladder showed up.<span style='mso-spacerun:yes'>  Including a picture and info seems futile.

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> 10022009 </b>

<p class=MsoNormal>PCR:   In order to optimize the reaction (and yield useable product) this reaction focused on optimizing the MgCl2 concentration in each reaction tube.<span style='mso-spacerun:yes'>

<p class=MsoNormal>The concentration gradient was:

<p class=MsoNormal style='margin-left:.5in'> 0.5&#956;M, 1.0&#956;M, 2.0&#956;M, 3.5&#956;M, and 5.0&#956;M – 6 reactions.

<p class=MsoNormal>The theory behind this was that purchased genomic DNA was preserved in TE buffer. It was a thought that the EDTA of the buffer was chelating all of the free magnesium ions in the reaction. By working with a gradient, it may be possible to use a specific concentration in further reactions.<span style='mso-spacerun:yes'>  Exp003 was modified: the new annealing temp was 60°C, hoping to make the reaction more stringent (preventing non-specific binding).

<p class=MsoNormal>Two different primer sets were used.<span style='mso-spacerun:yes'>  “New” primers were developed without the <span class=SpellE>biobrick extensions, as well as the mutagenesis primers. The PCR reaction was done in four parts: Colony PCR w/ primers “1/6” and “5/7” for point mutagenesis, as well as with new primers to amplify only the gene of interest.<span style='mso-spacerun:yes'>  Genomic DNA was used with the same primers. This created 2 reactions for each set.

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='text-decoration:none'> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> 10052009 </b>

<p class=MsoNormal>Gel Electrophoresis: 6 gels were run…two of which yielded banding in promising places.<span style='mso-spacerun:yes'>  It seems that the mutagenesis primers are giving good results for the smaller fragment. However, the other gels were not worth reporting.<span style='mso-spacerun:yes'>

<p class=MsoNormal>Results:

<p class=MsoNormal style='margin-left:.5in'><b style='mso-bidi-font-weight: normal'>Figure 5:</b> Banding seen is from Colony PCR and Primers 5/7.<span style='mso-spacerun:yes'>  It reveals the smaller fragment of the <span class=SpellE>OprB gene. The MgCl2 concentration that was optimal was 2.0&#956;M

<p class=MsoNormal style='margin-left:.5in'><b style='mso-bidi-font-weight: normal'>Figure 6:</b> Banding here is similar to the banding in the previous gel. However, the higher concentrations of MgCl2 (3.5 and 5.0&#956;M) showed brighter banding. This is in congruence with the previous hypothesis that the DNA that was preserved in TE buffer needed additional MgCl2 to overcome the chelating agents found in the EDTA.

<p class=MsoNormal style='margin-left:.5in'>

<p class=MsoNormal>

<p class=MsoNormal>

<p class=MsoNormal>

<p class=MsoNormal>

<p class=MsoNormal>

<p class=MsoNormal>