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From 2010.igem.org

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==Notebook==

June 14, 2010
In Attendance: Tom, Duke, Patrick, Robert, Steve, Gary, David, Ryan

3 Separate transformations attempted: <partinfo>pSB1C3</partinfo> [1-3A] (ChlorAmphenacol resistant), <partinfo>pSB1K3</partinfo> [1-5A](Kanamicin resistant), <partinfo>pSB1T3</partinfo> [1-7A] (Tetracycline resistant) Control Group: PET-17B (still had separate resistance for Ampicillan) Tom will be culturing some adjacently resistant control groups for our next trial, thereby eliminating the extra variable in our testing.

Procedural experience:
Transformations using 1-3A 50uL EXPERIMENTAL
200uL EXPERIMENTAL
250uL NEGATIVE CONTROL
Cells were plated and put in the 37C incubator. Plates labeled in RED.

9 Parts for PCR - 7 Distributed amongst us. Various participants were asked to look up their function and various bp lengths.

Patrick - <partinfo>Bba_R0010</partinfo>
Gary - <partinfo>Bba_R0063</partinfo>
Robert - <partinfo>Bba_J04450</partinfo>
Dave - <partinfo>Bba_R0062</partinfo>
Steve - <partinfo>Bba_J23009</partinfo>
Miles - <partinfo>Bba_I731014</partinfo>
Ryan - <partinfo>Bba_I13507</partinfo> - Intermediate Screening Plasmid - 861 bp

PCR for part [http://partsregistry.org/wiki/index.php/Part:BBa_R0063 R0063]:
Vial labeled with RED X is the EXPERIMENTAL REACTION
Vial labeled with RED Y is the TEMPLATE CONTROL
Vial labeled with RED Z is the PRIMER CONTROL

PCR Polymerase Chain Reactions

Requirements
1. Template
2. 2 Primers (limiting)
3. dNTP's (limiting)
4. Polymerase (limiting by heatcycle) Using Taq which tends to transcribe more errors.
5. Buffer (mg++) (limiting)

Primers are 18-30 bp long. Have to be at least 15 bp long. Primers bind to template DNA, one primer for each (5'/3' strands). Review: What is a base..? Sugar with A, C, T or G is a nucleotide. BP are the matching set of 5' and 3' hydrogen bonded nucleotides.

Polymerase links free dNTP's to the opened strand edges of basepairs.
Denaturation 95 - 98 degrees c... DNA Melts
Anealing 40 - 65 degrees c... Primer Binds
Extension 72 degrees c... Strands Extend
30 second cycles...

As the DNA heats it denatures then as it cools the primers anneal and polymerization takes place creating twice as many strands. The strands are held together only by hydrogen bonds, making it very easy to melt and reform. The polymerase acts like a little machine stitching the free dNTP's into the template.

BioBrick plasmids have bb prefix and suffix. Primer binding sites vf2, vr. Theoretically any area can become a primer site.

With PCR we can identify and amplify particular strands/compositions. You have to know how much of the requirements to use. Gary asked if Tom could show us where to find the optimized volume information at, and he agreed to pull out the research to show how to optimize.

With PCR you can also design primers and introduce mutations. If primers are too short they can bind to the wrong spot. Actual reactions may vary based on bp length and combination. Usually they are tweaked for the optimal over a series of trials.

PCR Reactions (Specific to this evening's trial)

	Experimental Reaction	Neg. Template Control	Neg. Primer Control>
Template	2 uL	0	2 uL
Forward Primer [VF2 310 uM]	16uL**	16uL**	0
Reverse Primer [VR 345 uM]	14uL**	14uL**	0
dNTPs	1uL	1uL	1uL
Rxn Buffer [5X-k]	10uL	10uL	10uL
Enzyme	0.5uL	0.5uL	0.5uL
Water	6.5uL	8.5uL	36.5uL
Total Volume	50uL	50uL	50uL
** indicates 1:100 dilution

34mM of dried DNA in primers ($12.50)

Several companies provide these primers: invitrogen is an example.

34 x 10-9 moles / 100 x 10-6 liters = 3.4 x 10 -7 moles/liter

Tomorrow we will look at the parts we pcr'd and then see how the bp length matches. If nothing happens its probably a pipetting error. If too many bands it may be an annealing issue.

We will also look at the cells we plated of the various transformed parts, 3A, 5A, 7A, and see again if any of the colonies grew.

---

June 10, 2010
In attendance: Duke, Gary, Robert, Patrick, Ryan, Miles, Steven, Liz, and Colin.

Tonight we tested the hypothesis that the plasmid did not code for the antibiotic resistance/that we did not use enough DNA. A Restriction Digest reaction was set up on the ligation reactions completed on June 7. The reactions were cut with EcoR1 and Pst1. The reactions were allowed to incubate at 37 degrees C for one and a half hours. The digests were then ran on a electrophoresis gel. No DNA was present as there was not enough DNA, thus proving Duke's hypothesis.

---

June 9, 2010
In attendance: Duke, Tom, Ryan, Patrick, Lisa, Steven. Missing: Kyle and Friend(Forgot his name)

1) Registration for iGEM site & Team affiliation, required to update team wiki.

2) Access and update wiki.. Consider stylization, as well as information content. Who is our audience? Team info - blurb and caption of team members. Couple of sentences about you and your interests. Lab Notebook? - Openwetware connection.

3) Research biobrick parts by accessing in Registry of Standard Parts. XF to see if new combination are already on file? If not begin documentation of the 3 new assemblies, with image using standard BioBrick icons. Document bp length of new parts to compare with Gel-Electrophoresis. How do we test efficacy? (Part for PPM still in brick.)

Is the media okay, is the antibiotic right. Positive tells you whether the cells themselves were capable of taking up our DNA. (Ideally, they should use the same antibiotic resistance, as our parts.

Negative groups tells us whether the antibiotic and media was effective and that the cells were not resistant to the media.

We know that cells were competent they were able to be transformed, and the antibiotic was effective. So the remaining question is whether they were able to be transformed.

The ligation's are in question. Generally the restrictions and ligation's go smoothly.

Tom's Hypothesis is questioning whether the media plates may have been mis-labeled with the wrong antibiotic resistance. Since we used a different resistance for controls, there was no telling.

Possible denaturing of enzymatic proteins from the heat shock on the initial restriction? 80c kill cycle. Vector ? - Linear plasmids - was the amount to small...? Parts - size already was confirmed, according to Tom

No Transformations.... so now what? Primers still on the way. Don't need to go to scratch.

The Core enzymes needed to perform the biobricks constructions... Spe1 pst1 ecor1 xba1 t4dna ligase polymerase

Project idea:
Polymerase in e.coli, tag, perhaps other enzymes could be self-manufactured/purified.

Basic tools/measurements

• Micropipetters [Measuring volume]
  
• Measuring Mass
  
• DV/HD camera - you tube videos and components.
  
• Mini-preps. Growing the cells and isolating DNA. Cell competency Preparations.
  

Lac polymerase - DIY-GEM

How do you purify?

What do you do with it... what is the process to create a project?

Hardware only takes you so far, these 5 basic tools. Restrictions Ligations Transformations/Plating PCR Gel Electrophoresis

The core will be good technique with these processes and then the ability to understand the existing database navigation with the proper questions to yield an experiment of interest.

Research educational tools from the MIT educator on IGEM site. Should we have an e-mail/Comments section added to wiki - openwetware, so observers can ask us questions?

---

June 8, 2010

In attendence: Colin, Patrick, Liz, Ryan, Duke and Tom. Missing: Andy, Scott.

So tonight we came in and took the 4 ligation's/new part combinations from yesterday and transformed them into the competent cells, using a heat-shock transformation, while also preparing the Control groups (one with nothing/one with the ampicillin resistance). 3 of the parts were Tetracycline resistant, and 1 was Chlorephenecol. After the 90 minute transformation cycle we plated 6 versions of each of the 3 parts along with 1 plate of control and one negative group.

The 6 plates were done in a 0, -1, -2 dilutions in 2 concentrations one of 50 ul, and one of 200 ul.

They will be left for tomorrows team to run gel electrophoresis to determine whether they have the appropriate combination links.

---

June 7, 2010

In attendance: Colin, Patrick, Robert, Ryan, David, Gary, Miles, Duke and Tom. Missing: Melissa.

Tom greeted us with 4 separate sheets that contained 3 reactions each for us to begin restrictions and began heating to let the enzymes cut. A lesson in what not to do, was offered as we began the heating cycle of restriction in the PCR blocks and he timing had been set to 35 seconds instead of 35 minutes, after which it heated to a kill cycle of 80 degrees and we had to reapply the enzymes, in case the enzymes had been denatured.

We had 6 individuals building the 4 sheets, 2 in redundancy.

The legend for the various parts is as follows...

1PO	<bbpart>BBa_R0063</bbpart>
2PO	<bbpart>BBa_P0412</bbpart>
3PO	<bbpart>pSBIT3</bbpart>
R (black marker)	<bbpart>BBa_R0062</bbpart>
I (black marker)	<bbpart>BBa_I13507</bbpart>
S (black marker)	<bbpart>pSBIT3</bbpart>
F (green marker)	<bbpart>BBa_F2620</bbpart>
I (green marker)	<bbpart><BBa_I13507</bbpart>
S (green marker)	<bbpart>SBIC3</bbpart>
R10 (green marker)	<bbpart>BBa_R0010</bbpart>
462 (green marker)	<bbpart>BBa_I0462</bbpart>
IT3 (green marker)	<bbpart>pSBIT3</bbpart>
A/R10 (green marker)	<bbpart>BBa_R0010</bbpart>
B/462 (green marker)	<bbpart>BBa_I0462</bbpart>
C/IT3 (green marker)	<bbpart>pSBIT3</bbpart>

Patrick cut parts <bbpart>BBa_r0063</bbpart> and <bbpart>BBa_p0412</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>.
Robert cut parts <bbpart>BBa_R0062</bbpart> and <bbpart>BBa_I13507</bbpart>, and the plasmid backbone <bbpart>pSBIT3</bbpart>.
Collin/Miles cut parts <bbpart>BBa_F2620</bbpart>, <bbpart>BBa_I13507</bbpart> and the plasmid backbone <bbpart>pSBIC3</bbpart> (labeled w/green marker).
The next step is to ligate them.

After which we had a round table discussion about what kind of projects we may follow up with and the process of using the NCBI databases to discover pre-existing sequence information related to our various ideas. One idea we have discussed was the option of creating a smoother introductory curve for fellow DIY-Bio commmunity members and the creation of home-brewed enzymes that might be to pricey for the amateur scientists. We ended the night with the beginning of the various ligation reactions, as seen above.

• Ligation Reaction for R10+462+IT3 in 'LIGATE 05X
• Ligation Reaction for A/R10+B/462+C/IT3 in 'L/LIG
• Ligation Reaction for F2620+I13507+IC3 with "Green Asetrisk"
• Ligation Reaction for R0062+I13507 in "Squiggly Sigil"