| pEXPR 1-pLac:mKate-2 | pEXPR TRE:Syk-pTet:RFP-TEVp | pEXPR TRE:Syk-pTet:RFP-tTEVp | Notes | ||
|---|---|---|---|---|---|
 |  |
|  |   | all three tTEV constructs are correct. TEV #3 is correct |
| tube name | Constants Definition | 2-log ladder | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR 1-pLac:mKate-2 | pEXPR TRE:Syk-pTet:RFP-tTEV | pEXPR TRE:Syk-pTet:RFP-tTEV | pEXPR TRE:Syk-pTet:RFP-tTEV | pEXPR TRE:Syk-pTet:RFP-TEV | 2-log ladder | pEXPR TRE:Syk-pTet:RFP-TEV | pEXPR TRE:Syk-pTet:RFP-TEV | pEXPR TRE:Syk-pTet:RFP-TEV |
| Tube Number | 1/1 | 1/2 | 1/3 | 2/1 | 2/2 | 2/3 | 1/2 | B1 | B2 | B3 | C1 | C2 | C3 | C2 | |||
| gel lane | 0 | 1/1 | 1/2 | 1/3 | 1/4 | 1/5 | 1/6 | 1/7 | 1/8 | 1/9 | 1/10 | 1/11 | 1/12 | NaN | 2/1 | 2/2 | 2/3 |
| DNA mass desired (ng) | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 | 500 |
| total volume(ul) | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
| DNA concentration (ng/ul) | 332.8 | 253.5 | 358.6 | 360.9 | 253.5 | 245.9 | 294.3 | 280.5 | 338.5 | 213.9 | 254.8 | 213.9 | |||||
| Enzyme Name | NheI | NheI | NheI | NheI | NheI | NheI | undigested | PvuII | PvuII | PvuII | PvuII | PvuII | PvuII | undigested | |||
| Buffer Name | 2.1 | 2.1 | 2.1 | 2.1 | 2.1 | 2.1 | water | 3.1 | 3.1 | 3.1 | 3.1 | 3.1 | 3.1 | water | |||
| gel/lane | 0 | 1/1 | 1/2 | 1/3 | 1/4 | 1/5 | 1/6 | 1/7 | 1/8 | 1/9 | 1/10 | 1/11 | 1/12 | NaN | 2/1 | 2/2 | 2/3 |
| DNA volume | #DIV/0! | 1.50 | 1.97 | 1.39 | #DIV/0! | #DIV/0! | 1.39 | 1.97 | 2.03 | 1.70 | 1.78 | 1.48 | #DIV/0! | 2.34 | 1.96 | 2.34 | |
| buffer volume | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| enzyme volume | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| strip tube # | 1/1 | 1/2 | 1/3 | 1/4 | 1/5 | 1/6 | 1/7 | 1/8 | 1/9 | 1/10 | 1/11 | 1/12 | 2/1 | 2/2 | 2/3 | 2/4 | |
| water volume | 17.0 | #DIV/0! | 15.5 | 15.0 | 15.6 | #DIV/0! | #DIV/0! | 15.6 | 15.0 | 15.0 | 15.3 | 15.2 | 15.5 | #DIV/0! | 14.7 | 15.0 | 14.7 |
| gel/lane | 0 | 1/1 | 1/2 | 1/3 | 1/4 | 1/5 | 1/6 | 1/7 | 1/8 | 1/9 | 1/10 | 1/11 | 1/12 | NaN | 2/1 | 2/2 | 2/3 |
| loading dye | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
| results | good | bad, pZDonor | good | bad pick | bad pick | bad | good | good | good | ??? | contaminated | good | |||||
| notes |
SDM
SDM 2,3, and 4/5 were run.
4/5 resulted from a mislabelling issue in which one sample is referred to by both numbers 4 and 5. This is one sample, not two, and no other samples are titled SDM 4 or SDM 5.
PCR Conditions:
SDM2 - Correcting CD79B Point Mutation from PCR - QuickChange Mutagenesis Q5 protocol 5 min elongation time, 70C annealing temperature
SDM3 - Correcting CD79B Point Mutation from PCR (Same as above, different protocol) - Golden Gate Mutagenesis Q5 protocol 5 min elongation time, 67C annealing temperature
LR
hEF1a:CymR
hEF1a-CuO:mKate
CMV5-Cuo:mKate
(pDEST_Seq4-Seq5)