Daily Lab Notebook
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Week 1: 6/10/09 - 6/12/09
First official meeting of the summer: finalizing the project details, setting up a tentative schedule.
We spent the first meeting narrowing in on the system we would use for our upcoming projects. After comparing the bacterial light sensing system with that of the modified yeast 2-hybrid system a consensus was reached to attempt the yeast system first. The yeast phytochrome based system seemed more sensitive and the bistable light inducible system would give us an added element of regulation, in that the system could be rapidly reset using IR wavelengths. This would be useful in a "bio-blackboard" configuration.
As a backup though, we should be prepared to revert to the bacterial system.
The potential problem for the yeast system is that phycocyanobilin (PCB) needs to be exogenously supplemented. It may need to be extracted from a suitable (algae?) source or the bacterial PCB biosyntheis pathway may need to be cloned into yeast, if possible. Also, if using yeast as a "sender" with red-luciferase, the luciferin substrate for luciferase also needs to be added externally. Uptake should occur if the media is made acidic.
David pointed out a more recent intein system that also utilizes the same Phytochrome/PIF3 domains that the modified yeast two-hybrid system uses.
We carefully studies schematics of the various light responsive genetic systems, including an attempt at adding an "inverter" component into the  ,
 from a few years ago, and the system created by  .
Week 2: 6/15/09 - 6/19/09
Lab Meeting Notes.
DB3.1
LB absorbance and scattering? What did the old IGEM team dilute in?
Intein people had a modification to the PCB extraction protocol—look that up.
PCR the genes we need out of a cDNA library.
Churchill lab has everything?—ask them for Arabadopsis cDNA library.
Arabadopsis 2 hybrid library
Email Woody Hastings? Fluorescent dinoflagellates! Shake them to read by.
Assembly of low-promoter from oligos.
DTT is a reducing agent, ATP is energy, required for various enzynmes in the system to work.
First step is to take phosphorylate the resuspended oligos. The 5 prime ends need to be phosphorylated so that they can be ligated. Oligos normally come without the five prime phosphate.
Mix equimolar amounts of the four oligos and label oligo mix, 20 uL of each oligo.
Use PCR tube.
20 uL H20
6 ul oligo mix
4 ul 10x PNK buffer
4 uL DTT
4 ul ATP
2 ul PNK
Total: 40 uL…….keep on ice.
Samples put in thermocycler. See protocol for program, called “Assembly”.
Tuesday
For obtaining the PhyA, PhyB, and PIF3 genes, we have a number of methods we are pursuing.
1. Obtain cDNAs from another lab
a. Tuesday—The Mathews lab is willing to give us PhyA, but does not have PhyB or PIF3. They can help us with the construction of the PhyB and PIF3. We should ask them if they have a protocol they follow….
b. Wednesday--I have emailed the Mathews lab back, and will hopefully be meeting with Sarah Mathews later this week.
2. Obtain cDNAs from Biobricks registry
a. Tuesday--Pulled PhyB and PIF3 from the registry, as well as the PCB synthesis genes. The PCB synthesis genes look good according to registry sequence, but PhyB and PIF3 cite sequencing irregularities, so I am not hopeful.
b. Wednesday—Oliver compared the sequences from NCBI to the sequences given in the BioBricks registry for the PhyB and PIF3 and it does not look like they are correct, so that is not promising. We will continue with growing up the PCB biosynthetic enzymes though.
3. Obtain cDNAs from a cDNA library
a. Tuesday—No one seems to have a sample of the cDNA library we can have. Asked in the lab plant kid from 261r was in, he’s checking for us but is not hopeful. Purchase of a cDNA library is too expensive, $1200)
4. Obtain Arabidopsis plant and make a cDNA library, and from that clone out the genes of interest
a. Tuesday—The Pierce lab has kindly given us two lovely Arabidopsis plants, and we have ordered the necessary kits and mortar and pestle to do the RNA extraction. Tomorrow must design primers for sequencing and for cloning the genes out…these can be multipurpose.
b. Wednesday--
5. Obtain a library from TAIR. Arabidopsis.org
a. Wednesday—We are going to order libraries from the Arabidopsis people. They are very cheap, $5 a line, with $125 shipping fee total per order, so it’s fairly cheap. Shipping is combined for all orders.
b. 4 different libraries ordered.
Initial email to Mathews
Hi Dr. Mathews,
My name is Amrita Goyal, and I am a member of the Harvard iGEM team. We are looking for the cDNA for the PhyB, PhyA, or PIF3 components of the light-activated signalling system in plants. We were wondering if you had these genes available in a suitable vector that we could obtain. Our goal is to subclone these genes into a yeast-two-hybrid system. We would really appreciate it if you had a sample available that one of our teammates could pick up.
Also, if you have an extraction protocol for phycocyanobilin, or know someone who does, we would also appreciate that information. Thank you so much!
Sincerely,
Amrita Goyal and the Harvard iGEM team
Mathew’s Response to me
Hi Amrita,
Sorry for the delay in responding -- I've been doing jury duty.
We can help with a PHYA cDNA clone, in either pUC 18 or a bluescript vector. We don't have PHYB or PIF clones, or an Arabidopsis cDNA library. But depending on your timeframe, I could help with PHYB and PIF clones.
For a phycocyanobilin extraction protocol, I recommend you get in touch with someone in Clark Lagarias' lab (cc'd on this message). Their web page is at: http://www.mcb.ucdavis.edu/faculty-labs/lagarias/
We may have some phytochromobilin, but we have no phycocyanobilin.
Let me know when you'd like to pick up some PHYA clone, and I'd love to hear a bit more about your experiments.
Cheers,
Sarah
My response to Mathews
Hi Professor Mathews,
I hope that jury duty went well!
If we could get the PHYA cDNA in the pUC18 vector from you that would be fantastic. We would very much appreciate your help with making the PHYB and PIF3. We acquired a couple of Arabidopsis plants from a grad student in Naomi Pierce's lab yesterday and we were planning to do an RNA extraction and clone the PHYB and PIF3 out ourselves, if you think that is the best way to proceed.
If you think the phycochromobilin would work, we would also appreciate a sample of that. We did get Spirulina and are planning to attempt a PCB extraction soon.
What we are planning to do for our iGEM project is to create a cellular blackboard, using the Phy system in yeast. We want to use the PhyA and/or PhyB to turn on transcription of a green luciferase, so we can "write" on the blackboard using a red laser pointer. We would then "erase" the blackboard with far-red light to turn off luciferase transcription (although we will just have to wait for the luciferase to degrade then).
We were also thinking of engineering a system where we have two populations of yeast cells communicate with eachother using light. We would turn on production of a red luciferase in one population by shining a laser on it. The light produced by that population would then be used to turn on production of the luciferase in another population of cells.
If you have any thoughts or advice on either of these projects please let me know!
I can come by any time today or after lunch tomorrow, let me know when is most convenient for you! I actually have a couple of questions for you if you have a few minutes to talk in person in the next couple of days. Let me know what your schedule looks like!
Thanks,
Amrita
Response from Lagarias, jclagarias@ucdavis.edu
Colleagues,
I have been contacted for a related request by a Wash University iGEM Team. I told them that we would have to complete an MTA to send any plasmids (and we do not have PIF clones that we are at liberty to send; they were obtained from other labs). FYI, I have attached a protocol for PCB isolation.
Best.
Clark Lagarias
PREPARATION OF PHYCOCYANOBILIN Lagarias Lab Method
3E-phycocyanobilin isolation: Lagarias Lab Method (Terry, MJ, MD Maines, and JC Lagarias. 1993. Inactivation of Phytochrome-Chromophore and Phycobiliprotein-Chromophore Precursors by Rat Liver Biliverdin Reductase. J. Biol. Chem. 268(35):26099-26106): 3E-phycocyanobilin (PCB) was prepared from lyophilized Spirulina platensis (Sigma) using a method similar to that described by Beale and Cornejo (Beale & Cornejo (1991a) J. Biol. Chem. 266, 22328-22332; Beale & Cornejo (1991b) J. Biol. Chem. 266, 22333-22345).
1. Spirulina powder was rehydrated in deionized water (30 ml/g dry weight) for 10 min.
2. The slurry was centrifuged at 30,000 x g for 20 min and the deep blue supernatant was decanted.
3. Phycocyanin was precipitated from this supernatant with 1% (w/v) TCA by incubation for 1 h at 4oC in the dark and then collected by centrifugation at 30,000 x g for 20 min.
4. After washing with methanol (2 x 20 ml/g Spirulina powder), the blue pellet was resuspended in methanol (2 ml/g Spirulina powder) containing 1 mg/ml HgCl2.
5. Following incubation for 20 h at 42oC in darkness, the protein was removed by centrifugation at 10,000 x g for 10 min.
6. 2-Mercaptoethanol (1 ul/ml) was then added to precipitate the dissolved mercuric ion, which was also removed by centrifugation (30,000 x g for 10 min).
7. The crude bilin mixture was then diluted 10-fold with 0.1% (v/v) trifluoroacetic acid and applied to a C18 Sep-Pak cartridge (Waters-Millipore Corp., Milford, MA). The Sep-Pak cartridge was sequentially washed with 0.1% (v/v) trifluoroacetic acid (2 x 3 ml) and acetonitrile/0.1% trifluoroacetic acid (20:80; 2 x 2 ml) followed by elution of the crude bilin mixture with 3 ml acetonitrile/0.1% trifluoroacetic acid (60:40) and drying in vacuo.
8. Spectral assay of the crude bilin mixture is performed at this point (use a 1/100 dilution) and a typical yield of 4 umol phycocyanobilin per 6 g dry weight Spirulina can be obtained.
9. This crude phycocyanobilin is quite pure but can be further purified by C18 reverse phase HPLC using a Varian 5000 liquid chromatograph and a Beckman Ultrasphere ODS column (4.6 x 150 mm; 5 um particle diameter). The solvent system used was ethanol/acetone/water/acetic acid, 19:14:66:1 (v/v/v/v) with a flow rate of 1.5 ml/min, and the column eluate was monitored at 370 nm (Cornejo et al, 1992).
10. HPLC-purified 3E-phycocyanobilin was concentrated using a C18 Sep-Pak as described above, dried in vacuo and stored at -20oC. Before use, phycocyanobilin was dissolved in dimethyl sulphoxide to a concentration of 1-1.5 mM.
11. An aliquot of each stock solution was diluted 200-fold into 2% HCl/methanol to estimate the bilin concentration spectrophotometrically. The molar absorption coefficient of 47,900 M-1 cm-1 at 374 nm for 3E-phycocyanobilin (Cole, WJ, DJ Chapman, and HW Siegelman. 1967. The structure of phycocyanobilin. J. Am. Chem. Soc. 89:3643-3645) were used for these determinations.
1. HO1
a. BB—726 bp, 4425 bp.
2. PycA
a. BB—750 bp, 4425 bp.
3. PhyB
a. BB—1863 bp
b. TAIR—3996 bp
4. PIF3
a. BB—302 bp
b. TAIR—1790 bp
Truncated is better
There is a truncated PhyA
PhyB is main mediator of low fluence red light, conversion to Far form. W PhyA, main mediator of responses to far red light, in shaded environments. 2 modes—responses to prolonged exposure to far red light, and low fluence exposure if PhyA totally held in the dark. Still converts w red. Low fluence response does not matter on quality of light—any small exposure to visible light.
PhyA will bind PIF3, but in plantae, never. Getting phyA into nucleus FI1 needed for translocation to nucleus. Nuclear localization signal.
Lagarias—has been trying to get both phytochromobilin and the phytochrome to express concurrently in yeast, most people use the bacterial for heterologous expression. Clarke would be the person to ask to see hwat he has gotten to work. –email him he will respond.
They have primers for all Phy B and could design primers for just truncated section. Try just amplifying from genomic DNA in a single exon. Def try from genomic DNA.
Probbablyjust want to work with PhyB. In some ways could be advantageous s to work with Phy A—differs in how sensitive it is to light—once it sees light it degrades rapidly. Goes into proteosome degradation, and maybe a proteosome box is exposed? Maybe that’s just specific in Arabidopsis. It’s an E3 ligase. We need to look at specific structure of the protein. In plants PhyA builds to high levels in the dark and disappears in light—degradation and turning down. In yeast dunno how it would behave. –maybe working with a couple or three just to make sure one is robust would be good.
Def worth trying both PhyA and PhyB—espworking with full length. Don’t know if truncated has same sensitivity characteristics. PhyA is most sensitive.
You need to express the protein in the dark. The trick is the conditions under which you express the protein—use minimum minimum green light. Just put in green filters, 25 w bulb. If you can do that….The person collecting the data has a spectroradiometer and he had built a red and far-red filter to push them between the red and far red films in the cuvettes. She will see if they still have that instrument. The trick will be the conditions.
Mathews: Primer sequences to help get useful fragments—send messages to truncated clone people
You will never convert everything back from Pfr. 3 fctors effecting rate—it will dark revertat a specific rate, you can push it back with far red, and then temperature—revert faster if pulsed with far red.
We need the vectors for the yeast-two hybrid system, and the strains of yeast they used. We are particularly looking for the strains that have the UAS driven reporter if you have them.
Yeast two hybrid system
Strain missing Gal4 and Gal80. Gal4 has DNA binding domain and activation domain. Binds to several promoters, one is pGAL1. There is also a GAL2 and GAL4. The strain usually has these two deletions, and those are redulated under galactorse, truned on by it. In the absence of galalctose it is blocked by Gal80. Usually in one of the GAL promoters you have GAL4 deleted and GAL80 deleted. You usually have a reporter, and you can measure by adding OMPG or whatever substrate. The strains come in two mating types, A or alpha. They are haplopd, when opposite mating types meet they fuse.
PRS303—0 means it integrates. You put in your gene of interest and you cut this plasmid in the selectable marker and linearize the plasmid and it integrates into the yeast genome and there is enough homology so youhave a good copy of the leucine gene. Andyou can have it driven under whatever promoter.
PIF3—FL
PIF3—Partial
PhyB—Partial
We need full length Gal4—this will
PCB questions--
Do we need to HPLC it? Will it work without it?
Can we just insert the genes into the yeast and have it express the enzyme?
Resources
www.addgene.org
www.atcc.org
www.sgd.org
www.arabidopsis.org
Week 3: 6/22/09 - 6/26/09
Discovery that there is a split luciferase, which works on the order of seconds. The concept would be to attach fragments to PIF3 and PhyB and then
Blackboard
Red light district
Signal transmission wire
C-terminus was tagged with phyB (phyB–YFPC). from Phee paper.
red mutant (S286N) luciferase . From wiki.
Renilla Luciferase
http://partsregistry.org/Part:BBa_J52008
Firefly Luciferase
http://partsregistry.org/Part:BBa_I712019
Use of a split-luciferase reporter to indicate interaction of PhyB and PIF3. I propose that for our system, we tag the PIF3 and PhyB with luciferase to form a split luciferase reporter system. When PhyB is stimulated with red photons, it undergoes a conformational change which gives it the ability to bind to PIF3 (phytochrome interacting factor 3). In order to reduce the amount of time between “writing” and “erasing” our theoretical blackboard, we had initially considered using a destabilized luciferase, but that did not solve the problem of
The theory behind a split-luciferase reporter. This technique was developed to help study protein-protein interactions in vitro, in vivo in cell culture, and in vivo in animals (like mice). The basis of a split luciferase reporter system is that when luciferase (like many other proteins, including GFP and ubiquitin) is cut in half, and each half fused to a protein of interest, the two halves lack enzymatic activity. However, when they are brought into close enough proximity to one another they reconstitute the functional enzyme. The split luciferin has been engineered to have minimal nonspecific affinity between the two halves, so it should only associate upon interaction of the two proteins the halves are fused to. Thus, upon photoactivation of PhyB, the binding of Pif3 and PhyB should facilitate the reconstitution of the functional luciferase, in the presence of the correct luciferin, result in a fluorescent signal.
Timing of luciferase reconstitution. Use of a split-luciferase system will allow us to turn on and off fluorescence in basically real time, without the delay of transcription to turn on the luciferase and degradation to turn it off. Studies have shown that association and dissociation occur within minutes. The split-luciferase reporter system has the distinct advantage over split-GFP systems that reconstitutiton of the luciferase is reversible, while reconstitution of the GFP fluorphore is irreversible. The GFP system is also disfavorable because GFP requires a high amount of excitation energy, and has high background, thus obscuring the signal (Cissell et al, 2009).
Luciferase species choice. There are several options for which luciferase we can use, including firefly luciferase, Renilla luciferase, and Gaussia luciferase.
Firefly luciferase is from the firefly (beetle), Photinus pyralis, and is 61 kD. It uses luciferin in the presence of oxygen, ATP, and Mg2+ to produce bioluminescence in the range of 550-570 nm (greenish yellow) (http://www.promega.com/paguide/chap8.htm#title2). When Luker et al examined the kinetics of cell lysates they found an increase in fluorescence with a t1/2 of less than one minute. They found it necessary to use cell lysates to examine kinetics of the interaction of the two halves because the presence of the cell membrane was preventing the rapamycin (the chemical they were using to induce association of the fused proteins) (Luker et al, 2004). Thus I assume that given that light will instantly permeate the cells, that we will see kinetics similar to those in the lysates. This paper does not mention dissociation kinetics.
Renilla luciferase is a 36 kD protein from the sea pansy (Renilla reniformis), which uses coelenterazine and oxygen to produce blue light, 480 nm (Promega). The issue with use of Renilla luciferase in animals is that the blue light does not penetrate tissues well, and that the coelenterazine is transported by the multidrug resistance P-glycoprotein, which according to Luker et al, as well as others, can cause problems with delivery (Luker et el, 2004).
Gaussia luciferase is derived from the marine copepod Gaussia princeps. This 19.9 kDa protein catalyzes a reaction involving oxygen and coelenterazine to produce bioluminescence at a peak of 470 nm. At 185 amino acds this is the smallest of the luciferases (Verhegen et al, 2002).
Luciferase fragment choice. Firefly luciferase. Luker et al found an optimized pair of firefly luciferase fragments as X --Nf Luc 2-412, and CfLuc 398-550 – Y. This was reduced from the initial as X -- Luc 2-435, and Luc 21-550 – Y, a significant decrease in overlap which presumably served to reduce constitutive activity of the N terminal fragment. In the paper by Luker et al, they found that “FRB-NLuc and CLuc-FKBP plus rapamycin produced a maximal bioluminescence of 2x 106 photon flux units per 1 x 104 cells in a 96-well format (7 x 104 photon flux units per ug of protein), 1,200-fold greater than untransfected cells or blank wells (Fig. 1E). By comparison, a control plasmid (pGL3) expressing intact luciferase produced 3-fold greater bioluminescence (6 x106 photon flux units 1 x 104 cells transfected with 33 ng of DNA). (Luker et al, 2004).”
In their 2007 paper Paulmurugan identified (NFluc 398/CFluc 394) as the ideal pair, claiming it to be an improvement upon existing combinations. This combination has according to their paper near-zero background signal from self-complementation. They claim this to be a direct improvement upon the N416-C398 pair from the Luker paper. In terms of fragment orientation they recommend Nfluc-FRB/FKBP12-CFluc, but this is only with one protein pair. They mention reduction in enzymatic activity when you attach other proteins to the N terminus of the protein, but mention that Luker had success with a protein on the N terminus of the N terminal fragment. (Paulmurugan et al, 2007).
Luciferase DNA sources. Renilla and firefly are in the BioBricks database, and we have received Firefly luciferase in red and green variants from a source. The red luciferase contains a single point mutation, and the green luciferase contains three mutations which shift it greener.
Fusion protein construction. All combinations are as follows:
Nfluc-PhyB,
Cfluc-PhyB,
PhyB-Nfluc,
PhyB-Cfluc,
Nfluc-PIF3,
Cfluc-PIF3,
PIF3-Nfluc,
PIF3-Cfluc.
Based on the suggestion that fusing things to the N terminal of luciferase reduces function, we should only attach proteins to the C terminal of the Nfluc. This leaves us with several possible pairs,
Nfluc-PhyB/C-fluc PIF3
Nfluc-PhyB/ PIF3-Cfluc
PhyB-Cfluc/ Nfluc-PIF3
Cfluc-PhyB/ Nfluc-PIF3.
Then the next question is do w e want to put the N or C terminus on PhyB or on PIF3? According to Phee, the cDNA of phyB or PAPP2C was subcloned into BiFC vector containing either the Nterminal region of YFP (yellow fluorescent protein) for YFPNE:: PhyB or the C-terminal region of YFP for YFP-CE::PAPP2C) (Phee et al, 2008). The Phee paper and Subramanian paper both put the protein fragment on the N terminus of PhyB, which leaves us with the following.
Nfluc-PhyB/C-fluc PIF3
Nfluc-PhyB/ PIF3-Cfluc
Cfluc-PhyB/ Nfluc-PIF3
Based on the following: Subramanian used the following BRET pair: RLuc-PhyB:PiF3-YFP (Subramainan et al., 2006), we should probably follow the same orientations.
Nfluc-PhyB/ PIF3-Cfluc
Red-shift of reconstituted proteins. Another factor that must be taken into consideration is the red shift known to result from reconstitution of the protein. In the paper by Cissell et al., they found that the emission max of the reconstituted Renilla luciferase was 495 nm, while native luciferase has a peak at 485 nm, a shift also seen with GFP. They describe this shift being a function of the change in proximity of amino acids around the chromophore as compared with native Rluc, and the presence of the negatively charged DNA which in these experiments drove the association (Cissell et al, 2009). I assume this may also be an issue with the firefly luciferase.
Lack of yeast studies. Split luciferase complementation assays have primarily been done in mammalian cells lines (HEK293T notably), and I have not seen a paper where they have been done in yeast. I saw one paper in which it was done in Arabidopsis. I see no significant reason it cannot be used in yeast, given that the only requirements are that the two fragments are in the same cellular compartment, and that luciferase be able to function in its native state in yeast, which it can. Also, appropriate luciferins must be provided. This may pose a problem with the coelentarazine, because of the problems mentioned with multidrug resistance peptide P? Luciferin will also be an issue because you need low pH for it to be taken up, but we can examine the use of esterified luciferins. Upon further examination the study of esterified luciferins was done in mammalian cells and in bacteria, but not in yeast. Further searches yielded no information about use of esterified luciferins in yeast (Craig et al, 1991). It appears that luciferase is not as commonly used in yeast as LacZ.
Conditions for luciferin uptake. We need to use D-luciferin. Addition of luciferin was done in the following manner: “D-luciferin (1 mM, 100 μl) in 0.1 M Na citrate buffer (pH 3.0 or 5.0) was pipetted into thewells containing induced cultures. The plate was briefly shaken and then immediately measuredusing a Victor multilabel counter (Perkin-Elmer Wallac, Turku, Finland) in the luminescence mode, using 1 s counting time. The light emission levels are expressed as RLU (relative light units = luminescence value given by the luminometer) and the normalized luminescence was calculated by dividing the RLU value of the induced culture by that of the blank solvent” (Leskein et al, 2003).
Peroxisomal targeting of luciferases. Insect luciferases contain a peroxisomal targeting signal at their C terminus, Ser- Lys-Leu (Leskein et al, 2003). In firefly luciferase this sequence is at the extreme C terminus, residues 548-550 (Gould et al, 1989). Leskein found that removal of the peroxisomal targeting signal resulted in better cell growth, which implies that peroxisomal targeting causes problems with cell growth. Also, use of the modified luciferase allows adequate luciferin uptake at pH 5 as opposed to pH 3 (Leskein et al, 2003). In that paper they had the luciferase under the control of a copper-inducible promoter. Modified luciferase was found to have 2 orders higher expression levels than wild type luciferase. This modification eliminates the need for centrifugation and resuspension of cells. They simply removed the peroxisomal targeting codons.
Positive control.
SNF1 and SNF4—they are in the same strain but if they are different sizes?
What is on drosophila library interacter? Taheyen—ask her for maps.
Negative control.
Nfluc/Cfluc
Specifications for Primers
Need to add appropriate restriction sites to the ends of the luciferases
Need to remove peroxisomal targeting sequence
Need to be in frame in pCETT
Tuesday June 23, 2009
PIF3 from biobricks matches first 300 bp of Arabidopsis PIF3 with the exception of one basepair. Changes glutamic acid to aspartic acid.
PhyB from biobricks matches first 300 bp of Arabidopsis PhyB.
PCB Subproject
PCB extraction—crude→HPLC?
Reconstitute pathway (HO1 and PcyA)—spec assay. Conditions?
PIF3 and PhyB Project
PIF3—APB (100 aa), Full length
PhyB—N Term (have sequences and primers)
Biobricks PhyB N
PIF3 APB
Library PhyB N
PIF3 APB
PIF3 Full
Plant PhyB N
PIF3 APB
PIF3 Full
July 1, 2009 (Weds)
We did the mRNA extraction from the Arabidopsis.
Primers were diluted to 100 uM, which was followed by a subsequent 1-10 dilution to make a total of 100 uL of each primer at 10 uM. Final reactions were .6 uM, which means we added 3 uL of each primer per rxn.
Dilutions
A. F.PIF3.SaII 242 uL
B. R. PIF3.partial.BsshII 214 uL
C. R.PIF3.FL.BsshII 208 uL
D. F.PIF3.BamHI.pACT2 240 uL
E. R.PIF3.partial.Xho1.pACT2 201 uL
F. R.PIF3.full.Xho1.pACT2 220 uL
G. F.PhyB.SpeI 170 uL
H. Nterm.BcII 213 uL
Reactions were mixed as follows:
27 uL RNAse free water
10 uL Buffer from kit
2 uL DNTPs
3 uL Forward primer
3 uL Reverse primer
2 uL RNA
2 uL Enzyme
Total: 50 uL
Reactions were run according to the following
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