Stella+M.


 * Week 3 & 4 **

Stella, good but include your Nanodrop images. Dr. B 090913 Friday 9/6--
 * Week 1 & 2 **

__Agarose Gel:__

Ladder failed to show due to a punctured well, and PCR will need to be repeated. Figure 1: Agarose gel results of PCR of pgbr22 plasmid showing distinct bands on wells 2-4, corresponding to samples A, B, and C, respectively, but with no ladder for comparison due to the first well being punctured.

Monday 9/2 -

__Nanodrop Practice:__

Objective was to determine the concentration of pgbr22 plasmid. Graphic results located in lab book (will upload graphs 9/9)

The concentrations recorded were low on average, possibly due to contents settling in the tube. Due to the low average, I used Imran's average concentration for determining amount of plasmid to be used for DNA sequencing and PCR.

__DNA Sequencing:__

Sample of pgbr22 was submitted and returned, but then I received a notification that it was run again. Final results are:

NNNNNNNNNNNNNNNNNNNNNCNNGNNNNGNNNNNNCCNTNNNNNCGNNNNNNNNNNNNNNGNGNNNCTCANNGNGGNNNNATGNNNNNNGCGANNNNNNGNGNNGAGNANAGNNANACCNCCNNNNTNNACGNTCGNGNNCNNANNNNNNNTNAANACTTCCANTNNNNNNNNGCCNANCNNCCGTTNCNCNANNNNTTTGGANGCNGNCTNNNTNTNNNNTNGNNATGCGNACTTGCNGNANAGNTGTNTCACTTTNCAANAATGNAGCCNTANACNNTTNNCCANGTNCCNTNNNNGANNCNNNCNNCNNNGCANCCCGTNNGGTTCCCTGCCNNGTGGAGGCTNNNGNNTNNNANGNNNANNNNNCNGAANNNTNNGNAACANATNTTGTCNCTNTANATGANCTCNNTTNNCNNGGATNGTGNNNNCNTTGNNNNNNNTACNCNANGCANANNCNTTACGTANNNNGCNTGACANGGNAGATNCNNNNNGGACTGACNGNTNNNNTAACCNNGGATGTCTNCNTGGTNAANTNNNNAAAGANNNNNCNNNNNNNNGGGANGGNGCNNNTNNANTNNNNNNNTCNNNNNNCNGANGTNNNNNNNNNNGNCNNCTGTNGNNACNNANTGTGNNNNNNTNNNCNGNNNN

Where N's are base-pairs unable to be read by the sequencing machine. Due to the large number of N's, and the fact that the first sequencing failed, the sample must have been contaminated in some way.

__Oligo Design__:

Primers for phosphoatase 2C from //T. gondii://

30 Oligos:

1 ATGGCGGACGCTAAGACCCTGCTGGGCAAGGTTAAACGTGCGACGGGTATGGGTG 55

2 GCGGTGTATTTCGGTTTTTTTGCTACAGAAGGACCTTCACCAACACCCATACCCGTCGCA 60

3 CAAAAAAACCGAAATACACCGCGACCGTTCCTGGCTTCACCCCGCCATCCGGTGATCAGC 60

4 ATAAACTCGCCGGACGTGTCAACAGCCATAAATTCGTTCATACGCTGATCACCGGATGGC 60

5 CACGTCCGGCGAGTTTATGCGCCACCTCTACATCGAAGAGGGCCGTACCGTGTGTGCGTC 60

6 GTGCGGAGACTCAGAGGTTGGACGACGATTGCGAGAGGTCGCAGACGCACACACGGTACG 60

7 ACCTCTGAGTCTCCGCACTCTGATGACGTTGTTGTGGTAGAAGGCATGCTCCGTGGCCGT 60

8 CGACCTTGGAAACCGTCGAACATAGCGTGAACACGGGTTTCCGGACGGCCACGGAGCATG 60

9 CGACGGTTTCCAAGGTCGCCATTCTGCTATGTGGCTGGCCCAGAACGTAATGAACTACCT 60

10 GGGTGATTTCTTCCTCGTTCACGTCGCGGAGATCGTTCAGGTAGTTCATTACGTTCTGGG 60

11 TGAACGAGGAAGAAATCACCCGTCAGTTCGAGCGCATGGACGGTGACCTCCGTGCAGCAA 60

12 TAACGAACGAAAATGATGAGCGCAGAGCTGCCGCCAGGCAGGTTTGCTGCACGGAGGTCA 60

13 CGCTCATCATTTTCGTTCGTTACGAGAAAAAGCCGACCGAAGCGCGTGTAGTTGGTCGTC 60

14 CGGCCACAGAGGTGAATTCCTTCGCACCCTCCGGAACGATTTGACGACCAACTACACGCG 60

15 GAATTCACCTCTGTGGCCGAGGCACTCGGTGGTCCGCTGATGCCGGTTGTTGCAATGAAT 60

16 ATGGTGTAAATACCTTTCGCAGCGCGAGGGTCACGACGGAAATTCATTGCAACAACCGGC 60

17 CTGCGAAAGGTATTTACACCATTCATGTTGCGTCTCTGGGTAACTCTCGTTGCGTTCTGA 60

18 TATGCGGGGTAGACAGGTGGATCGCAGTGCGACCAGATTTCAGAACGCAACGAGAGTTAC 60

19 CACCTGTCTACCCCGCATACCGCGTCTTCTCACAAAGAACGTCACCGCGTTCAAGCAGCG 60

20 CCGCCGAGGAGGAGTTCGCCGTTAACAGTGGTAAACACACCACCCGCTGCTTGAACGCGG 60

21 GAACTCCTCCTCGGCGGTGTTGTACCGATGACCCGTGCGTTCGGCTCTTTCGACTTCAAA 60

22 AGAAACCAGGTCTTGTTGGAGCTTACCTTGGCCACCTTTTTTGAAGTCGAAAGAGCCGAA 60

23 CTCCAACAAGACCTGGTTTCTGCGGTTCCAGACGTTACCACCTTCTTTGCGTATCCTGGT 60

24 AGTGCGCAAAGGCACCCGCGGTACCTGCTACGATGTCGTCACCAGGATACGCAAAGAAGG 60

25 GGTGCCTTTGCGCACTTCCGCTCTCATGCCGCGATTGCAGCCGCTATTGCGCTGTACCCG 60

26 TACCATGGCCTTGGCGGCGTCCAGAACCGTTTCAGGAGATACCGGGTACAGCGCAATAGC 60

27 CCGCCAAGGCCATGGTAGTCAATGCCAAACGTCGTAAAGTTACCAAAAACATCTCTACGT 60

28 AGAACGAGTACGAGACTCCGGCAGATGGCGAACAAACGTAGAGATGTTTTTGGTAACTTT 60

29 CGGAGTCTCGTACTCGTTCTCAGAAAATGCTGGAGGGCACGAGCGGCGAGAACGGTGAGT 60

30 TTACTCACCGTTCTCGCCG 19

__PCR:__

PCR was run with pgbr22 and stored in -20 degree freezer until Friday