Research+Page-+Indra

=Indra's Research=

Week 14:

 * Completed Enzyme and Vary Substrate Assays on YopH protein
 * Cleaned lab
 * Turned in final Research Report
 * Turned in Lab Notebook

Week 12-13:

 * Attempted ten times to complete Protein Expression Protocol
 * Determined that pNIC-Bsa4 clone had become linearized and was not the correct length (determined by gel check)
 * [[image:Gel_Check_Linear.jpg width="400" height="300" caption="Fig. 1: Image of Agarose gel that determined pNIC-Bsa4 clone was linearized. "]]

Week 11:

 * Ran secondary job in HF9 Library, got good fitness scores in the high 80's
 * Ran first job in cb_306 library, did not get any good fitness scores so did not proceed with a secondary job
 * Ran first job in CB-Kin library, got good fitness scores in high 70's and low 80's. Running secondary job this week
 * Completed Materials & Methods for Virtual Screening paper
 * Began Protein Expression Protocol.

Week 10:

 * Ran first job in HF9 Library, set up secondary run, but due to the lack of licenses it has not been able to run yet
 * Set up first run for CB_306 Library, but due to the lack of licenses it has not been able to run yet
 * Completed Materials and Methods for Cloning paper

Week 9:

 * Created a Homology Model for //Shigella flexneri// using// Escherichia blattae // acid phosphatase as a model. Had 93% similarity to //Shigella flexneri// apyrase
 * [[image:IndraShukla_HomologyModel.png width="512" height="384" caption="Fig. 1: Homology Model overlap of 1D2T (green) and Shigella apyrase (blue)"]]
 * Got results back from DNA Sequencing, BLAST'ed sequences against Codon Optimized Sequence and determined that cloning was successful
 * Proceed into Expression with IS4-for - **Indra - good deal. Be sure to note that you will have to transform it into BL21(DE3) cells first. Also grow up some in DH5alpha and do miniprep to keep an archival sample of your vector. - Dr. B**
 * Started Virtual Screening, ran first job
 * Used ICM to minimize //Shigella// apyrase locally and make it usable by GOLD

Week 8:

 * Grew up Master Plate successfully.
 * Had growth in each of the 8 tubes that were grown up over night
 * Miniprep samples that were grown up over night
 * ==[[image:vdsstream/master_plate.jpg width="320" height="423" caption="Fig. 1: Master Plate of colonies. Area 1 was voided because I accidentally threw away the toothpick used to transfer the colonies"]]==
 * [[image:IS1.jpg width="800" height="493" caption="Fig. 2: Nanodrop of my first sample, IS1"]]
 * [[image:IS2.jpg width="800" height="495" caption="Fig. 3: Nanodrop of IS2"]]
 * [[image:IS3.jpg width="800" height="495" caption="Fig. 4: Nanodrop of IS3"]]
 * [[image:IS4.jpg width="800" height="495" caption="Fig. 5: Nanodrop of IS4"]]
 * [[image:IS5.jpg width="800" height="495" caption="Fig. 6: Nanodrop of IS5"]]
 * [[image:IS6.jpg width="800" height="495" caption="Fig. 7: Nanodrop of IS6"]]
 * [[image:IS7.jpg width="800" height="495" caption="Fig. 8: Nanodrop of IS7"]]
 * [[image:IS8.jpg width="800" height="495" caption="Fig. 9: Nanodrop of IS8"]]
 * Took samples to DNA Sequencing.
 * Took samples to DNA Sequencing.

Week 7:
> == ==
 * Completed the Transformation and Annealing step of pNIC-Bsa4 cloning protocol
 * Set up Master Plate using Master Plate protocol and 8 tubes for samples
 * ==[[image:vdsstream/VDS_10.jpg width="320" height="423" caption="Fig. 1: Tube A of Transformation and Annealing Step. There were not as many colonies on this plate"]]==

Week 6:

 * Finished Virtual Screening and PyMol Refresher
 * Prepared pNIC-Bsa4 as an accepting vector
 * Attempted to complete the Transformation and Annealing step of the protocol, but failed



Week 5:

 * Nanodrop samples from PCR on 9/25/11, got a concentration of 7.8 ng/uL
 * Ran the 1st PCR in Protocol for pNIC-Bsa4 cloning off of Cloning Vector with updated PCR protocol. I used a concentration of approximately 75 ng/uL of my. I also added more cycles to the PCR protocol, increased the number from 20 to 30 additional cycles.
 * Nanodrop samples from PCR with 75 ng/uL concentration. Moving on to transformation with this PCR product.
 * Prepared pNIC-Bsa4 as Accepting Vector. Nanodrop samples, did not gel check.
 * [[image:vdsstream/VDS_1.jpg width="400" height="294" caption="Fig 1: Nanodrop results from 9/25/11. Concentration was too low, so I had to run the protocol again."]]
 * [[image:VDS_3.jpg width="400" height="562" caption="Fig 2: Gel Check of PCR that had 75 ng/uL of plasmid and had 10 additional cycles on PCR."]]
 * [[image:VDS_2.jpg width="400" height="327" caption="Fig 3: Nanodrop of PCR product with 75 ng/uL of plasmid"]]
 * [[image:VDS_4.jpg width="480" height="260" caption="Fig 4: Nanodrop results of Preparation of pNIC-Bsa4 as Accepting Vector"]]

Week 4:

 * Ran the 1st PCR in Protocol for pNIC-Bsa4 cloning off of Cloning Vector with updated PCR protocol. I used the concentrations stated in the protocol
 * [[image:NEWPCRFAIL.jpg width="400" height="300"]]
 * Ran the 1st PCR in Protocol for pNIC-Bsa4 cloning off of Cloning Vector with updated PCR protocol. However I used different concentrations than what the protocol stated.
 * [[image:IUSPCR3.jpg width="400" height="300" caption="Fig 4: Gel check of updated PCR. I used very high concentrations of pUC-19 vector (110 ng/uL). "]]

Week 3:

 * Ran Gel Check for 1st PCR in pNIC-Bsa4 cloning off of pUC-19 again. However this was not successful because lines were not in correct place due to the fact that the PCR protocol was off.
 * [[image:IndraPCRpNICBsa4.2.JPG width="384" height="288" align="left" caption="Fig. 2: Gel Check of PCR for pNIC-Bsa4 cloning. This time, it was deduced that the wrong PCR protocol was being used."]]

Week 2:

 * Ran PCR for cloning for pNIC-Bsa4 cloning off of pUC-19
 * [[image:pNIC-Bsa4_Cloning.jpg width="400" height="348" caption="Fig 1: Gel Check of PCR for pNIC-Bsa4 Cloning. Unfortunately the PCR failed, possibly because I took too long to complete the process"]]
 * Prepared pNIC-Bsa4 as Accepting Vector.
 * Remade forward primer

Transformation Efficiency Lab:






PCR Gel: pCherry Results


Indra - any thoughts on why the PCR is failing? - Dr. B

Oligonucleotides (Primers):
1 ATGAAAACCAAAAACTTCCTGCTGTTTTGCATTGCGACCAATATGATCTTCATCC 55 2 GGTGAGGAAACCTTCTGCTTTCAGGGCGTTCGCAGACGGGATGAAGATCATATTGGTCGC 60 3 AAAGCAGAAGGTTTCCTCACCCAGCAGACCTCTCCGGATTCTCTGTCTATCCTGCCGCCA 60 4 CGCTTTGTCCGCCAGGAAAACAACAGAATCCTCCGCTGGCGGTGGCGGCAGGATAGACAG 60 5 CCTGGCGGACAAAGCGCACTACGAATTTGGTCGTTCTCTCCGTGACGCGAACCGCGTTCG 60 6 AAGGCGAGACCGAAGTTCTCGTAGTACGCATCTTCAGACGCCAGACGAACGCGGTTCGCG 60 7 AGAACTTCGGTCTCGCCTTCTCTGACGCGTACGGTATGGACATCTCTCGTGAAAACACCC 60 8 AGTCCTGCAGTACCTGGGTCAGCAGCTGGTACAGGATCGGGGTGTTTTCACGAGAGATGT 60 9 CCCAGGTACTGCAGGACTCTCATGACTATGCTGTTCGCAACGCTAAAGAATACTACAAAC 60 10 AGGTCGCGTCTTTGTAGATAACGAACGGGCGAACACGTTTGTAGTATTCTTTAGCGTTGC 60 11 TTATCTACAAAGACGCGACCTGCACCCCGGATAAAGACGAAAAAATGGCGATCACCGGTT 60 12 CGCAACCGCCCAACCGAAGCTCGCGTGACCGGACGGGTAAGAACCGGTGATCGCCATTTT 60 13 GGTTGGGCGGTTGCGCTGATCCTGGCAGAAATCAACCCGCAGCGTAAAGCGGAAATCCTG 60 14 CGCAGATAACACGAGATTCACCGAACTCGTAACCACGACGCAGGATTTCCGCTTTACGCT 60 15 GTGAATCTCGTGTTATCTGCGGCGCACACTGGCAGTCCGACGTTGAAGCGGGTCGTCTGA 60 16 AATTCCGGCGTGTTGTGCAGCACCGCAACTACAGACGCACCCATCAGACGACCCGCTTCA 60 17 GCACAACACGCCGGAATTCACCAAATCTCTGAGCGAAGCGAAGAAAGAATTCGAAGAACT 60 18 TTACGGGGTCAGTTCGTTGGTCGGAGTGTTCAGTTCTTCGAATTCTTTCTTCGC 54