Imran+Z.


 * Good, but could use more results. Show pictures of plates. -UM **
 * Week 10 & 12 **


 * Transformation of T. gondii PP2C CDS in pNIC to BL21(DE3) competent cells**

The miniprepped plasmid+gene sample of colony 8 was transformed into BL21(DE3) cells. 50 ng of plasmid were added to 50 ul of the competent cells and 250 ul SOC media. After transitioning from ice to 42C and back to ice, the mixture was incubated and shaken at 37C and transferred onto two LB+Kan plates. One plate housed 50 ul of the bacterial mix and the other plate housed 10 ul. Both plates were incubated at 37C overnight.


 * Submitting to DNA Sequencing **

To ensure that colony 4 and colony 8 had positive clones, the miniprep-purified samples of colony 4 and colony 8 were re-sent to the sequencing facility, this time with the opposite primer. The colony 4 pNIC sample was sent with pLIC-Rev and the colony 8 pNIC sample was sent with pLIC-For.

DNA sequence (subject, bottom) of the miniprepped Colony 4 pNIC+insert with pLIC-Reverse primer blasted against the PP2C gene CDS with forward and tail primers (query, top). 75% Query Cover, 98% Indentity.

DNA sequence (subject, bottom) of the miniprepped Colony 8 pNIC+insert with pLIC-Forward primer blasted against the PP2C gene CDS with forward and tail primers (query, top). 79% Query Cover, 98% Indentity.

The DNA sequencing facility released the sequences of each of the 8 colonies. It was found that the CDS sequences of colony 4 (pLIC-For) and colony 8 (pLIC-Rev) were potential positive clones.

DNA sequence (subject, bottom) of the miniprepped Colony 4 pNIC+insert with pLIC-Forward primer blasted against the PP2C gene CDS with forward and tail primers (query, top). 70% Query Cover, 99% Indentity.

DNA sequence (subject, bottom) of the miniprepped Colony 8 pNIC+insert with pLIC-Reverse primer blasted against the PP2C gene CDS with forward and tail primers (query, top). 78% Query Cover, 98% Indentity.

The 8 purified vector samples were submitted to the DNA sequencing facility. To samples of colonies 1-4, 4 ul of 2.5 mM pLIC-Forward primer was added. To samples of colonies 5-8, 4 ul of 2.5 mM pLIC-Reverse was added. All samples contained 300 ng of the purified plasmid with gene insert and a total volume of 12 ul.

Great analysis but missing image results and the corresponding captions. - BN
 * Week 9 & 10 **
 * Miniprep of pNIC-Bsa4 with T. gondii PP2C gene **

Each transformation tube was spun down to harvest the pellets. The miniprep process was performed on each sample of cells to purify them, and the resulting vector was eluted in 50 ul Elution Bufer.

The vector concentration from Colony 1 after miniprep was 69.8 ng/ul. The vector concentration from Colony 2 after miniprep was 56.3 ng/ul. The vector concentration from Colony 3 after miniprep was 65.2 ng/ul. The vector concentration from Colony 4 after miniprep was 97.7 ng/ul. The vector concentration from Colony 5 after miniprep was 83.3 ng/ul. The vector concentration from Colony 6 after miniprep was 78.2 ng/ul. The vector concentration from Colony 7 after miniprep was 71.8 ng/ul. The vector concentration from Colony 8 after miniprep was 87.6 ng/ul.


 * Picking Colonies of DH5a containing pNIC-Bsa4 holding the PP2C gene **

Plate 2 DH5a colonies (2 ul vector + 10 ul insert)

Plate 1 DH5a colonies (2 ul vector + 4 ul insert)

Both plates yielded colonies, and 4 colonies from each plate (8) were picked and allowed to grow in transformation tubes with 5 ml LB+Kan for 16 hours. Because the colonies were separated by ample distance, a master plate was not created. Instead, the colonies to be picked were circled and numbered directly on the plates.


 * Cohesive End Generation + Annealing & Transformation of the pNIC-Bsa4 vector and PP2C gene**

After this process, annealing of the vector and gene, in addition to transformation in DH5a competent cells, was carried out. This was done by creating two tubes: Tube A with 2 ul of the vector and 4 ul of the insert, and Tube B with 2 ul of the vector and 10 ul of the insert. 25 ul of DH5a competent cells were added to each tube. The tubes were incubated and the resulting bacterial solution from each was spread onto LB-Agar + Kan + 5% Sucrose plates, which were incubated for 1 day.

Cohesive end generation was performed on the PCR insert and cut pNIC accecpting vector individually. This process created sticky ends on both pieces such that the vector would readily accept the gene insert. T4 DNA Polymerase was used to catalyze DNA production.


 * Week 7 & 8** More Data? -UM


 * Cutting pNIC-Bsa4**

A 2 ul sample of the eluted cut vector indicated a concentration of 86.5 ng/ul.

A 2 ul sample of the eluted cut vector indicated a concentration of 83.8 ng/ul.

The purified pNIC vector was cut using the Bsa-I restriction enzyme in order to prepare it for acceptance of the gene. The PCR cleanup process was performed to purify the resulting cut vector.


 * Midiprep of raw pNIC-Bsa4 (not cut, no gene insert)**

After the midiprep process was performed on a new batch of cell pellets, the resulting vector concentration was 98 ng/ul - much higher.

The vector concentration after the initial Midiprep process was ~25 ng/ul, so it was decided that a fresh batch of competent cell pellets be re-midiprepped to gain a higher final concentration.


 * Obtaining unmodified pNIC-Bsa4**

A DH5a competent cell colony containing the raw pNIC vector was picked and grown in 160 ml LB + Kan. The resulting pellets were obtained by centrifugation.


 * PCR Cleanup of PP2C gene**

The Nanodrop analysis indicated a gene concentration of 225.5 ng/ul for the first trial.

The Nanodrop analysis indicated a gene concentration of 226.3 ng/ul for the second trial.

The samples used for the PCR Squared reaction were consolidated into a single tube. This tube was the reagent used for the PCR Cleanup process using the Sigma Aldrich EP008 PCR Cleanup Kit. The Nanodrop machine was initialized with nanopure water and blanked with Elution Solution from the kit. Nanodrop analysis indicated an average concentration of 225.9 ng/ul.

Good job on the analysis just do it separately from the caption. Good job with the progress and good luck on cloning! - Michael T.
 * Week 5 & 6**


 * PCR Squared**

The PCR Squared process was successful. 10 ul of sample from each 50 ul PCR tube was mixed with 2.5 ul 6X Loading Dye, of which 10 ul was loaded into each of 4 wells. In comparison to the 100bp ladder, a band is present at ~1200bp in each well, which is near the length of the targeted gene (1230bp). It is evident that the PCR Squared process was able to heavily copy the gene of interest. From this point, the cloning process can ensue.


 * "LB + Kan" Plate Preparation**

5 LB agar plates were prepared with ~20 ml of autoclaved LB solution in each plate, were treated with Kanamycin antibiotic, and stored.


 * Secondary PCR**



The secondary PCR process was successful on the initial trial. In comparison to the 100bp ladder, there is a distinct band present at ~1200bp, which is near the size of the targeted sequence. After addition of the primers, the gene length increased from 1203 bp to 1230 bp. The secondary PCR process was successfully able to bridge gaps between the oligonucleotide primers.


 * Week 3 & 4**


 * Forward & Reverse Tail Primer Design**

Ordered Forward: TACTTCCAATCCATGGCGGATGCTAAAACC

Ordered Reverse: TATCCACCTTTACTGTTATTCGCCGTTTTCACC



The DNA sequence snippet for Protein Phosphatase 2C from Taxoplasma Gondii. The forward primer is highlighted green, and the reverse blue. 18 base pairs are highlighted yellow succeeding or preceding the primers, respectively. This entire sequence was taken and inserted into the SacB region of the pNIC-Bsa4 accepting vector.


 * Primary PCR**



The third primary PCR trial was successful. In comparison to the first and second trials, the temperature for the annealing phase was dropped to 57C from 58C, and 2 ul of 10 mM dNTP was used as opposed to 1 ul of 10 mM dNTP. The visible smear represents the various sizes of the collection of primers present in the sample that have not been chained together. A 100bp ladder is shown for comparison.



The second primary PCR trial was unsuccessful. A 100bp ladder is shown.



The initial primary PCR trial was unsuccessful. A 100bp ladder is shown.


 * Oligonucleotide Mixture**

The oligo mixture was created by mixing 30 ul of primers (1 ul from each of the 30 primers) to 70 ul of autoclaved nanopure H2O to achieve a final volume of 100 ul.


 * Restriction Enzyme Digest - pgbr-22**





Compared to the virtual gel, it is evident that EcoRI and PvuII restriction enzymes cut where where specified. In the "EcoRI + PvuII" well, the smallest band is low and faint, but is present in the correct location based off of the virtual gel.

Imran, good - do you have another PCR result? If not - redo this PCR. Also, can put the sample ID in the Nanodrop field so that the image has the info - DR. B 090913
 * Week 1 & 2**


 * Plasmid (pgbr22) PCR**



The second PCR trial of the pgbr-22 plasmid indicated that the plasmid was successfully copied during the pcr process. Although there is some contamination in the Sample D well, there is a distinct band in each well at ~ 1000 bp, which is the size of the plasmid. Additionally, the band is much more distinct in the Sample C and Sample D wells, which is expected due to the higher concentration of plasmid used.

The gel image after the pgbr22 PCR process. There is a single distinct band in the Sample C well that aligns with the size of the pgbr22 sequence (~1000 bp).


 * Nanodrop pgbr22:**

The initial nanodrop run to determine the concentration of pgbr22 in the sample.

The second nanodrop run to determine the concentration of pgbr22 in the sample.

The averaged concentrations from run1 and run 2 indicated a pgbr22 concentration of 152.95 ug/ul in the sample.


 * Oligonucleotide Primer Design:**

SEQUENCE 1: PROTEIN LENGTH = 401

1 MADAKTLLGKVKRATGMGVGEGPSVAKKPKYTATVPGFTPPSGDQRMNEFMAVDTSGEFM 61 RHLYIEEGRTVCASATSRNRRPTSESPHSDDVVVVEGMLRGRPETRVHAMFDGFQGRHSA 121 MWLAQNVMNYLNDLRDVNEEEITRQFERMDGDLRAANLPGGSSALIIFVRYEKKPTEARV 181 VGRQIVPEGAKEFTSVAEALGGPLMPVVAMNFRRDPRAAKGIYTIHVASLGNSRCVLKSG 241 RTAIHLSTPHTASSHKERHRVQAAGGVFTTVNGELLLGGVVPMTRAFGSFDFKKGGQGKL 301 QQDLVSAVPDVTTFFAYPGDDIVAGTAGAFAHFRSHAAIAAAIALYPVSPETVLDAAKAM 361 VVNAKRRKVTKNISTFVRHLPESRTRSQKMLEGTSGENGEX

The initial protein sequence for T. gondii Protein Phosphatase 2C provided to DNA Works.

The DNA sequence # 1 is:

1 ATGGCCGACGCAAAAACTCTCCTCGGTAAAGTTAAACGTGCGACCGGTATGGGTGTTGGT 61 GAAGGTCCGTCCGTTGCTAAAAAGCCAAAATACACCGCTACCGTTCCAGGTTTCACCCCG 121 CCATCTGGTGACCAACGTATGAACGAATTCATGGCGGTTGACACCTCCGGCGAATTTATG 181 CGCCACCTCTACATTGAAGAAGGTCGTACCGTGTGTGCATCCGCGACCTCTCGTAATCGT 241 CGCCCGACGTCTGAATCCCCTCACTCTGATGATGTAGTTGTCGTTGAAGGTATGCTGCGT 301 GGTCGTCCTGAAACGCGTGTTCACGCGATGTTTGACGGTTTCCAGGGCCGTCATTCTGCG 361 ATGTGGCTGGCGCAGAACGTTATGAACTACCTGAACGACCTGCGCGACGTAAACGAGGAG 421 GAAATTACCCGTCAATTCGAGCGCATGGACGGCGATCTGCGTGCTGCCAACCTGCCTGGT 481 GGTTCCAGCGCACTGATCATCTTTGTTCGTTACGAAAAGAAACCTACCGAAGCCCGCGTT 541 GTAGGTCGTCAAATCGTTCCTGAAGGTGCGAAGGAATTCACGTCCGTAGCGGAGGCACTC 601 GGCGGTCCGCTGATGCCAGTTGTGGCGATGAACTTCCGTCGTGACCCGCGTGCAGCTAAG 661 GGTATTTACACCATCCATGTGGCGTCTCTGGGTAACTCTCGTTGCGTTCTGAAATCTGGC 721 CGTACCGCGATCCACCTGTCTACCCCGCACACTGCGTCCTCTCACAAGGAACGTCACCGC 781 GTTCAAGCGGCAGGCGGCGTGTTTACCACGGTTAACGGCGAGCTCCTGCTCGGTGGTGTT 841 GTGCCTATGACGCGCGCATTCGGTTCTTTCGACTTCAAAAAAGGCGGTCAGGGTAAACTG 901 CAGCAAGATCTGGTAAGCGCGGTTCCGGATGTAACCACTTTCTTTGCGTATCCGGGTGAT 961 GACATTGTTGCGGGCACCGCGGGTGCCTTTGCTCATTTCCGTTCCCACGCGGCTATTGCA 1021 GCCGCGATTGCGCTCTACCCGGTTAGCCCGGAGACCGTTCTGGACGCCGCGAAAGCGATG 1081 GTGGTCAATGCCAAACGTCGCAAAGTAACCAAAAACATTTCTACCTTCGTTCGTCACCTG 1141 CCGGAATCTCGCACGCGCTCTCAGAAAATGCTGGAAGGTACCAGCGGTGAAAACGGTGAA 1201 TAA

The DNA sequence for T. gondii Protein Phosphatase 2C as translated by DNA Works.

PARAMETERS FOR TRIAL 1 Total Size Of Gene ......... 1203 nt Protein Residues ........... 401 Mutatable Residues ......... 385 Fixed Nucleotides .......... 48 nt Degenerate Nucleotides ..... 0 nt Oligo Size ................. 60 nt Annealing Temp ............. 62 +/- 1*C Oligo Concentration ........ 1.00E-7 M Sodium Concentration ....... 5.00E-2 M Mg2+ Concentration ......... 2.00E-3 M Codon Frequency Threshold .. 10% Repeat Threshold ........... 8 nt Mispriming Threshold ....... 8/18 (6 exact) nt

Various information provided by the DNA Works analysis for the T. gondii Protein Phosphatase 2C sequence.

1 ATGGCCGACGCAAAAACTCTCCTCGGTAAAGTTAAAC 37 2 CGGACGGACCTTCACCAACACCCATACCGGTCGCACGTTTAACTTTACCGAGGAGAGTTT 60 3 TGGTGAAGGTCCGTCCGTTGCTAAAAAGCCAAAATACACCGCTACCGTTCCAGGTTTCAC 60 4 CGCCATGAATTCGTTCATACGTTGGTCACCAGATGGCGGGGTGAAACCTGGAACGGTAGC 60 5 CGTATGAACGAATTCATGGCGGTTGACACCTCCGGCGAATTTATGCGCCACCTCTACATT 60 6 ACGAGAGGTCGCGGATGCACACACGGTACGACCTTCTTCAATGTAGAGGTGGCGCATAAA 60 7 CATCCGCGACCTCTCGTAATCGTCGCCCGACGTCTGAATCCCCTCACTCTGATGATGTAG 60 8 GCGTTTCAGGACGACCACGCAGCATACCTTCAACGACAACTACATCATCAGAGTGAGGGG 60 9 GTGGTCGTCCTGAAACGCGTGTTCACGCGATGTTTGACGGTTTCCAGGGCCGTCATTCTG 60 10 GGTCGTTCAGGTAGTTCATAACGTTCTGCGCCAGCCACATCGCAGAATGACGGCCCTGGA 60 11 GTTATGAACTACCTGAACGACCTGCGCGACGTAAACGAGGAGGAAATTACCCGTCAATTC 60 12 GGCAGGTTGGCAGCACGCAGATCGCCGTCCATGCGCTCGAATTGACGGGTAATTTCCTCC 60 13 GTGCTGCCAACCTGCCTGGTGGTTCCAGCGCACTGATCATCTTTGTTCGTTACGAAAAGA 60 14 GATTTGACGACCTACAACGCGGGCTTCGGTAGGTTTCTTTTCGTAACGAACAAAGATGAT 60 15 GCGTTGTAGGTCGTCAAATCGTTCCTGAAGGTGCGAAGGAATTCACGTCCGTAGCGGAGG 60 16 CGGAAGTTCATCGCCACAACTGGCATCAGCGGACCGCCGAGTGCCTCCGCTACGGACGTG 60 17 TTGTGGCGATGAACTTCCGTCGTGACCCGCGTGCAGCTAAGGGTATTTACACCATCCATG 60 18 ATTTCAGAACGCAACGAGAGTTACCCAGAGACGCCACATGGATGGTGTAAATACCCTTAG 60 19 ACTCTCGTTGCGTTCTGAAATCTGGCCGTACCGCGATCCACCTGTCTACCCCGCACACTG 60 20 CGCCTGCCGCTTGAACGCGGTGACGTTCCTTGTGAGAGGACGCAGTGTGCGGGGTAGACA 60 21 GTTCAAGCGGCAGGCGGCGTGTTTACCACGGTTAACGGCGAGCTCCTGCTCGGTGGTGTT 60 22 CTTTTTTGAAGTCGAAAGAACCGAATGCGCGCGTCATAGGCACAACACCACCGAGCAGGA 60 23 CGGTTCTTTCGACTTCAAAAAAGGCGGTCAGGGTAAACTGCAGCAAGATCTGGTAAGCGC 60 24 CATCACCCGGATACGCAAAGAAAGTGGTTACATCCGGAACCGCGCTTACCAGATCTTGCT 60 25 TTTGCGTATCCGGGTGATGACATTGTTGCGGGCACCGCGGGTGCCTTTGCTCATTTCCGT 60 26 ACCGGGTAGAGCGCAATCGCGGCTGCAATAGCCGCGTGGGAACGGAAATGAGCAAAGGCA 60 27 ATTGCGCTCTACCCGGTTAGCCCGGAGACCGTTCTGGACGCCGCGAAAGCGATGGTGGTC 60 28 GAAGGTAGAAATGTTTTTGGTTACTTTGCGACGTTTGGCATTGACCACCATCGCTTTCGC 60 29 AGTAACCAAAAACATTTCTACCTTCGTTCGTCACCTGCCGGAATCTCGCACGCGCTCTCA 60 30 TTATTCACCGTTTTCACCGCTGGTACCTTCCAGCATTTTCTGAGAGCGCGTGCGAG 56

The 30 oligonucleotides that must be synthesized for T. gondii Protein Phosphatase 2C outputted by DNA Works.