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Team:Baltimore US/Notebook
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*Ligation Reaction for R0062+I13507 in "Squiggly Sigil" | *Ligation Reaction for R0062+I13507 in "Squiggly Sigil" | ||
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Revision as of 17:54, 17 June 2010
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==Notebook==
June 16th, 2010
In attendance: Lisa, Ryan, Tom, Colin (for a short time), Duke (for a moment)
Missing: Steve
Earlier in the day, Robert and Ryan performed a mini-prep of the pcr'd plasmid backbones 3A1(2) and 5A1(2).
Robert also began working on finding optimized conditions for pcr.
With the small turnout, Ryan began the digestion restriction with Colin's assistance. We combined the 2 strains of 3A1 and 5A1 together, then put together 3 - .8 eppendorf's with 50 ul of rxn ingrediates.
(2 tubes of positive control of both along with 1 negative control that contained no enzymes and 40 ul of H2O)
5 ul of DNA
1 ul of Pst
1 ul of EcoR1
5 ul of Buffer
38 ul of H20
50 ul - Negative Controls Labelled with minus sign.
After preparation, the cells were put into the pcr block for a 30 minute heat cycle.
Tom will be heading to Arizona, tomorrow, so we called it a night so he could pack and go.
It has been suggested that we run a "Low-Heat Agar Gel" so we can find and physically splice out the appropriate sized plasmid structures.
June 15th, 2010
In attendance: Patrick, Ryan, Duke, Tom, Steve
Missing: Colin, Liz, Scott, Andy
Tom says results seemed to have worked for all of the transformations this time. Some better then others.
Steve asks the question.. "Why did transformation work better this time?"
Tom discusses differences in attempts.
Plasmid contains part a, another contains part b, another part c is simply a linear piece of DNA representing a Tet resistant backbone with the EcoRi and PstI restriction sites.
We cut Part A with EcoR1 and Xba1.
We cut Part B with Spe1 and Pst1.
We cut the Linear Backbone Part C wit EcoR1 and Pst1.
Then we combined everything into one tube using 2 ul of each part into 1 tube, and added t4 DNA ligase, hoping to get a Part A joined to Part B, because the Spe1 combines with the Xba1. And the Part A would join Part C with the EcoR1 and the Part B with Part C at the Pst1 site.
(See photo of board - )
Last week, we got nothing, so why?
Where could things go wrong?
They could go wrong at the restriction digestion reaction (less likely as process is fairly robust, and had been tested the day previously.)
Ligation reaction may not have worked.. (again less likely, process is fairly robust). If the buffer isn't mixed well it may separate.
How do we test? we could perform pcr to see if the ligation took place, using primers to amplify the ligated segment checking with gel electrophoreis to identify.
Transformation reaction - (Except we ran positive controls successfully)
Still possible to have mixed up the media, since positive used Amp instead..
Tom checked the media and found that wasn't the case.
Amount of Plasmid may not have been accurate. (The only thing we didn't physically check ahead of time.)
Pure operator/Pipetting action - such as the 30 second heating followed by
kill cycle.
Detection limits for Ethidium Bromide 10-15 ngs of a product.
(3600 bp ) (650 gm/b.p molar weight)
2,340,000 Grams per mole of 3600 bp molecule.
50 nanograms
(50 x 10-9) / 2,340,000 = 2.1 x 10-14 moles
Transformation rxn is 1 x 10 (-8/-9), still fairly efficient.
We did 2 things last night, we did the transformations into the stocks..
By using assembly strategies that don't use the same resistance as the parts, we can more efficiently screen out non-transformed pieces.
Previously in Genetic Engineering all parts and pieces had variorus resistances where as bb tries to create a standard.
Cells can be lost in replication due to metabolic costs, and new generations lacking the Costly programming tend to outgrow the special cells.
When designing the assembly strategy try to use a plasmid backbone with a different resistance to allow the filtration/screening of parts that didn't transform correctly.
Gel-Electrophoresis Prep Review for tonights run.
Agarose 2 % for smaller parts will create a more viscous environment.
0.8% for the larger parts will allow longer molecules to move better.
50ml final volume
Weigh out the agarose (.4 gms for the 0.8%) (1 gm for the 2%)
Emphasize again the importance of NCBI databases... http://www.ncbi.nlm.nih.gov/
With links to BLAST, PubMed, and other official literature. Program called entree which links all the information together.
When reviewing the tubes from last evening left in PCR there was difficulty in identifying the tubes that were done by each individual. We spent a little time using the process of elimination. Knowing Patrick, Steve, Miles, Ryans and Gary's without issue, we had to guesstimate which was Roberts, and which was David's. The reason we needed differentiation was to create a loading template, in one of the two different agarose preparations.
June 14, 2010
In Attendance: Tom, Duke, Patrick, Robert, Steve, Gary, David, Ryan, Miles
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> - 200 bp
Gary - <partinfo>Bba_R0063</partinfo> - 151 bp
Robert - <partinfo>Bba_J04450</partinfo> - 1,069 bp
Dave - <partinfo>Bba_R0062</partinfo> - 55 bp
Steve - <partinfo>Bba_J23009</partinfo> - 97 bp
Miles - <partinfo>Bba_I731014</partinfo> 1,938 bp
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"
