IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/24

{| width="800"
 * style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]] iGEM iGarden
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Procedures
Today's objective is to obtain purified DNA of the arabadopsis allergen panel. The last attempted to PCR the allergen genes was unsuccessful, possibly due to the Phusion Polymerase warming up too much. This time, the reaction will be repeated and kept on ice throughout the entire procedure.

Once we obtained purified genes, we will proceed to digestion with Xba1 and Pst1, ligation into V0120 vectors, and transformation of the vector into E. coli.

Our procedures for today were...


 * 1) PCR of arabadopsis allergen panel
 * 2) Nanodrop of PCR products
 * 3) Electronic Gel of PCR products
 * 4) Purification of PCR products
 * 5) Nanodrop of purified genes

Our E. Gel did not run properly, so we decided to run a traditional gel of the purified PCR products. But because we wanted to minimize DNA loss, we ran another PCR reaction (PCR #2) of the purified DNA from the first PCR reaction (PCR #1). This way, we would only lose a small amount of purified DNA but have enough DNA to see the results clearly on a gel.


 * 1) PCR #2 of purified allergen genes
 * 2) Gel Electrophoresis of PCR #2 products

Results
PCR of arabadopsis allergen panel

Like last time, we followed the [Silver: PCR] protocol. This reaction was successful for all of the allergen genes. This reaction was kept on ice throughout the entire procedure, unlike last time's, and we feel that the temperature difference was key in the success of this reaction.

Nanodrop of PCR products

Our concentrations after PCR in (ng/μL) were:

Electronic Gel of PCR products

Nothing showed up on the E. Gel. Our imaging machine was not working properly and we could see very little other than blurry ladders. The ladders did not run straight, so there may be problems with our E. Gel. For example, the gel might have been expired.

Purification of PCR products

We used the [Qiagen PCR purification kit with microcentrifuge protocol]. We eluted with 30 μL of Buffer EB to keep the concentration of our end product as high as possible.

Nanodrop of purified genes See chart in Nanodrop of PCR products for results.

PCR #2 of purified allergen genes

We used 2 μL of purified DNA, leaving around 28 μL of purified DNA products for our digest, ligation, and transformation.

Gel Electrophoresis of PCR #2 products

A gel image of PCR #2 products showed that all lanes (other than the ladder) had DNA segments with lengths of 300bps. This means that purified DNA is contains the correct genes that the initial primers were meant to amplify.

Primers for Barstar, Barnase, and Gal4DBD arrive
Primers:

BB.Barstar.Fwd - 36.1 nM, diluted with 361μL H2O 5'- CCT TTC TAG AAT GAA AAA AGC AGT CAT TAA C -3'

BB.Barstar.Rev - 30.0 nM, diluted with 300μL H2O 5'- AAG GCT GCA GCGGCC GCT ACT AGT TTA AGA AAG TAT GAT GGT GAT GTC -3'

BB.Barnase.Fwd - 38.5 nM, diluted with 385μL H2O 5'- CCT TTC TAG AAT GAA AAA ACG ATT ATC ATG G -3'

BB.Barnase.Rev - 13.2 nM, diluted with 132μL H2O 5'- AAG GCT GCA GCG GCC GCT ACT AGT TTA TCT GAT TTT TGT AAA GGT CTG -3'

BB.Gal4DBD.Fwd - 38.5 nM, diluted with 385μL H2O 5'- CCT TTC TAG AAT GAA GCT ACT GTC TTC TAT -3'

BB.Gal4DBD.Rev - 32.9 nM diluted with 329μL H2O 5'- AAG GCT GCA GCG GCC GCT ACT AGT CGA TAC AGT CAA CTG TCT TTG -3'

Digestion
Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes. The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.

Gel Image, first digestion:

PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)



The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.

Yeast Growth Assay
To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition. Each beaker also contained y190 yeast strains and YPD medium.

To construct a growth curve of the yeast under the 0μM, 1μM and 10μM conditions, 1mL from each flask would be pipetted into a cuvette and measured in a spectrophotometer ever hour to assess yeast density.

Unfortunately in this first attempt, no growth was observed after 4 hours, in the control and experimental groups, so the test was called off, to be repeated tomorrow.