User:DrJones1935/19 July 2012

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I. Measure the Concentration of Miniprepped Plasmids

  • Bring samples and Buffer EB bottle upstairs to Take3 plate reader
  • Add 2 uL of each sample according to the following chart:
Blank (EB) Blank
Blank Blank
pBAV1K-T5-gfp I (15Jul12) pBAV1K-T5-gfp II (15Jul12)
pBR322 I (18Jul12) pBR322 II (18Jul12)
pBR322 III (18Jul12) pBR322 IV (18Jul12)
  • Use the program to blank the machine and measure sample concentrations
RESULTS

Concentration:

Plasmid Concentration (ng/uL) Approx. volume (uL) Approx. total (ug)
pBAV1K-T5-gfp I 280.432 ~46 12.90
pBAV1K-T5-gfp II 412.41 ~42 17.32
pBR322 I 1.748 ~500 0.874
pBR322-T5-gfp II 2.744 ~500 1.372
pBR322-T5-gfp III 2.738 ~500 1.369
pBR322-T5-gfp IV 1.749 ~500 0.875

II. Ordered Updated PCR Primers from Keck

  • Primers were ordered with the name/sequence in the following table:
Name Tm (Tm overhang/Tm anneal) Length (bp) Sequence (overhangANNEAL)
cT5_pBf 74.16 (68.90/56.06) 66 (46/20) 5'-tgactctagatttgctttgtgagcggataacaattataatagattcAATTGTGAGCGGATAACAAT-3'
cT5_pBr 79.55 (75.05/56.42) 60 (44/16) 5'-gtaactgcaggagagcgttcaccgacaaacaacagataaaacgaAAGGCCCAGTCTTTCG-3'
cT5_p2f_2 74.20 (69.30/55.65) 66 (46/20) 5'-aattgtgagcggataacaattactagagaaagaggagaaatactagATGACCATGATTACGGATTC-3'
cT5_p2r_2 72.60 (63.89/55.41) 50 (30/20) 5'-tctccatacctcatcactcctcaatattacTTATTTTTGACACCAGACCA-3'
cT5_p3f_2 70.57 (62.42/56.57) 54 (32/22) 5'-caaaaataagtaatattgaggagtgatgaggtATGGAGAAAAAAATCACTGGAT-3'
cT5_p4r_2 81.11 (76.12/57.88) 59 (44/15) 5'-aaggcccagtctttcgactgagcctttcgttttatttgatgcctTCAGGTCGAGGTGGC-3'
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