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User:DrJones1935/19 July 2012
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*See [[MAGE_in_Naturally_Competent_Organisms#cT5_.28normal.29|Project Page]] for more info | *See [[MAGE_in_Naturally_Competent_Organisms#cT5_.28normal.29|Project Page]] for more info | ||
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Latest revision as of 01:53, 4 October 2012
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' |
- See Project Page for more info
