ResearchPage+-+Jericho


 * Jericho' Research**

=WEEK 14=



Since my protein failed I had to do a phosphatase enzyme test on YopH. This showed pretty good results since there is an increase then a slight plateau towards the end.

It seems that my protein completely failed because it did not even show up on the FPLC graph. It is suppose to appear between the two peaks in the dashed blue line because it has a molecular weight of 35 kDa.

== My graph is showing a pretty good curve with a concentration of 1.17 mg/ml. This should work in the FPLC. = = =WEEK 13=

No results this week, still working on virtual screening on compounds to see my top ligands match the compound being tested in wet lab. = = =WEEK 12= Jericho - I think this looks ok. Not sure which is your Elution one lane, but you shoudl thinkg about going to FPLC to further purify. - Dr. B

The band in Elution 1 that seems like my protein is between 35 kda and 25 kda. The MW of my protein is around 35800. I may have failed my gel.



The MW of my protein is around 35800 and it seems by looking at Elution 1 that there is a band around that area. Elution 2 is almost invisible because it is not suppose to have much protein. Sample's 2 and 3 are all over the place because it is the soluble fraction and flow through,it has everything.

I chose DNA instead of Protein Nanodrop last week, so this week I uploaded the Protein Results.



==

Elution 1 has a higher concentration than Elution 2. This is how it is suppose to be because elution 1 has more protein than elution 2

= = =WEEK 11 - Jericho - do these Nanodrops using the Protein setting, not the DNA setting! - Dr. B=

Protein Characterization is the next step.

These are the concentrations I received for Elution 1 and 2.



Most of the protein was released after the first 5 ml of Elution Buffer. This may account for the high concentration.

==

This may have a lower concentration because most of the protein was released after the first elution, this second elution released the remaining protein.

=WEEK 10=

I am currently working on protein expression.

This are the top ten ligands for cb_306 and HF9. Since my target is PP2Ctg it has calciums in it's active site. I only left 6 waters that had a polar contact with the calciums when I screened both libraries. These have high scores since they are all over 120.

= = = = = = = = = = =WEEK 9=

I have to continue running more libraries against my target in order to find top ligand.

I will have to run my run with calcium's again, because I will add the waters that have a polar contact with the calcium ions.

This is the top ten ligands for my two runs. I made 1 run of my target with it's calcium ions and another run with no calcium's. An important note is that they both have the same top ligand.

=WEEK 8=

I am still working on the virtual screening of my target. I got a really good concentration for my secondary PCR after PCR2 and the cleanup process.



To make the secondary PCR I used a ul of the first primary PCR and then I just made a gel out of 100bp and the secondary PCR. I got a really strong band.

= = = = =WEEK 7=

I am currently working on my virtual screening of my target.



This is my 2nd Trial of Primer Overlap, and it failed since my secondary PCR did not work and it even seems that my primary PCR has a small smear.

This is my second nanodrop using the other 100 ul of PCR squared and I got a lower concentration.

This is my first nanodrop using 100 ul of the 200 ul of PCR squared. It gave me a really low concentration. A good concentration should be 300 ng/ul.

=WEEK 6= - good, -- Dr. B

PCR Primer Overlap VDS



The length of my gene or interest is 996 nucleotides long. The ladder shows that the stain is within that region (1000).

I believe my secondary PCR was successful because it has 1 stain in the 1000 region. The primary PCR shows a smear as well.

=WEEK 5=

I finished 3 protocols this week, they were PCR Primer Design for pNIC-Bsa4 Cloning, PCR Primer Design for Primer Overlap Assembly PCR, Virtual Screen Refresher.


 * PCR Primer Design for Primer Overlap Assembly PCR**

FINAL SUMMARY FOR 1 SOLUTION Tm - 62 Len - 60 Score - 0.000 TmRange - 2.0 Short - 16 Long - 60
 * 1) Olig - 24
 * 2) Repeat - 0
 * 3) Misprime - 0


 * PCR Primer Design for pNIC-Bsa4 Cloning**

Upstream: 5’ TACTTCCAATCCATGATGGATGTACCACCTACCATT 3’ Downstream: 5’ TTCTTCAAAAAAACCGACTAACAGTAAAGGTGGATA 3’ Reverse Complement: 5’ TATCCACCTTTACTGTTAGTCGGTTTTTTTGAAGAA 3’

Accepting Vector with Insert ....GGAGATATACATATG **CACCATCATCATCATCAT** TCTTCTGGTGTAGATCTGGGTACCGAGAACCTG **TACTTCCAA** **TC ** **C ** __ **ATG** GATGTACCACCTACCATTCACGTTCCTCTGCCGCCGACGTCTTACCCGGCGTTCGACGCGGCGATCTTCACCA __ __CATCGGTGGCCGTAAACA....GCGCCGACAACACCGCGATGACGGTTTTCTTCAAAAAAACCGAC** TAA ** __ **CA **  **GTAAAGGTGGATA** .....

The blue font is the 6 His Tag. The Purple font is the Forward and Reverse Primers, while the teal font is the start and stop codon.


 * Image of Accepting Vector**




 * Virtual Screen Refresher**

Top Ten Ligands
 * # Fitness S(hb_ext) S(vdw_ext) S(hb_int) S(int) time File name Ligand ||  ||   ||   ||
 * 94.41 9.13 71.64 0.00 -13.22 1032.578 'output_1_101/gold_soln_all_m1_3.sdf''BindingDB_50228029|CB5k_1all|sdf|5007|dock7' ||
 * 84.87 5.25 66.65 0.00 -12.02 451.885 'output_304_404/gold_soln_all_m376_5.sdf' '5193911|CB5k_1all|sdf|1795|dock10' ||
 * 84.77 12.42 58.67 0.00 -8.32 407.745 'output_405_505/gold_soln_all_m411_8.sdf' '5109057|CB5k_1all|sdf|298|dock1' ||
 * 84.70 12.35 58.39 0.00 -7.95 204.417 'output_203_303/gold_soln_all_m205_2.sdf' '5230391|CB5k_1all|sdf|2662|dock2' ||
 * 84.51 10.00 62.96 0.00 -12.05 403.548 'output_304_404/gold_soln_all_m304_2.sdf' '5185261|CB5k_1all|sdf|1521|dock10' ||
 * 84.47 20.00 55.59 0.00 -11.97 215.833 'output_405_505/gold_soln_all_m406_1.sdf' '5143470|CB5k_1all|sdf|906|dock9' ||
 * 84.12 24.47 50.24 0.00 -9.43 237.895 'output_304_404/gold_soln_all_m305_4.sdf' '5157370|CB5k_1all|sdf|1200|dock9' ||
 * 82.41 16.92 55.45 0.00 -10.75 822.580 'output_304_404/gold_soln_all_m327_1.sdf' '5159193|CB5k_1all|sdf|1238|dock5' ||
 * 81.80 1.67 60.20 0.00 -2.64 339.182 'output_102_202/gold_soln_all_m106_7.sdf' '5249855|CB5k_1all|sdf|3261|dock1' ||
 * 81.61 12.17 54.49 0.00 -5.49 347.381 'output_102_202/gold_soln_all_m107_8.sdf' '5251494|CB5k_1all|sdf|3310|dock10' ||
 * 81.61 12.17 54.49 0.00 -5.49 347.381 'output_102_202/gold_soln_all_m107_8.sdf' '5251494|CB5k_1all|sdf|3310|dock10' ||

=WEEK 4=

My experiment went better than last time for pLIC sequencing vectors of pNIC-Bsa4. In this case the highest concentration had the stain, but none of the others had any stain.
 * 2nd Trial of Protocol PCR Sequencing of pLIC**

=WEEK 3=

I also worked on my PyMOL Refresher this week.


 * 1st Trial of Protocol PCR Sequencing of pLIC**

This is the image for PCR protocol for pLIC sequencing vectors of pNIC-Bsa4. I failed it probably because of wrong dilutions.

=WEEK 2=

==
 * 2nd Trial of PCR of pGBR22**

Lane 9 has EcoR1 so there should be 1 band because EcoR1 only cuts once while in Lane 10, which is Pvu11, there should be 2 bands because Pvu11 cuts twice.


 * This is the Submit to DNA Sequencing**



This is the Forward Sequence for the pGBR22 using nucleotide collection as the database.



This is the Reverse Sequence for the pGBR22 using nucleotide collection as the database.

Using nucleotide blast, Human G+T, and the somewhat similar sequences option the Forward sequence for the pGBR22 I submitted was attempted to be found. It seems my sequence had a low alignment score for almost everything.

Using the same options as the Forward sequence the pGBR22 sequence I submitted gave this. It seems it also has a low alignment score for almost everything since it has poor alignment for 80 percent for the nucleotides.

=WEEK 1=


 * This is the PCR of pGBR22**


 * 1st Trial of PCR**



My experiment failed, since the DNA did not appear on any lanes besides the 100bp ladder and lane 7 where you can see two faint stains. The mistake may have been when I was diluting the templates and the Taq DNA Polymerase NEB.