Digest and gel verification (hEF1a:LacI, TRE:mKate, pLac:mKate, hEF1a:CD79A-XTCSX-gal4VP16)
enzymes:
| Ladder | pEXPR 1-Hef1a:LacI-2 | pEXPR 1-TRE mKate-2 | pEXPR 1-pLac mKate-2 | hef1a:cd79A-3T3-gal4 | hef1a:cd79A-XTCSX-gal4 |
|---|---|---|---|---|---|
| ApaI | SalI | ApaI | BglII, XbaI | BglII, MluI | |
| 9 | 10 | 11 | 12 | ||
 |  |  |  |  |  |
| Ladder, TRE:mKate, pLac:mKate, hEF1a:LacI, 3T3, 3T9, 3T15, 6T6, 6T9, 6T12 | Ladder, 9T3, 9T6, 9T9, 9T12, 9T12, 9T15, 12T6, 12T9, 12T12, 15T3, 15T9, 15T15 | Notes: |
 |  | The LRed constructs (1st 3 lanes after the ladder, left gel) seem to be in the wrong order. The band pattern in lane 1may be the failure mode of TRE:mKate, but there is an extra band at around 10kb which doesn't make sense, also there seems to be some undigested plasmid as well. The band pattern in lane 2 is probably the correct hEF1a:LacI, even though it says it's pLac:mKate. The band pattern in lane 3 is probably the correct pLac:mKate, even though it says it's hEF1a:LacI In gel 2, all of the linker constructs have the correct band pattern, except that some of them have an additional band at around 4-5 kb. We sent 9TCS9 for sequencing, if it is correct we'll send the rest of them for sequencing. In gel 1, 3TCS3 and 3TCS9 have the correct band pattern. The rest also have the extra 4-5 kb band and an expected 1.5 band is not visible. |
Sent For Sequencing:
| Sample1 | Sample2 | Sample3 | Sample4 | Sample5 | Sample6 | Sample7 | Sample8 | Sample9 | Sample10 | Sample11 | Sample12 | Sample13 | Sample14 | Sample15 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| S1-9T3 | S2-9T3 | S4-9T3 | S1-9T6 | S2-9T6 | S4-9T6 | S1-9T9 | S2-9T9 | S4-9T9 | S1-9T12 | S2-9T12 | S4-9T12 | S1-9T15 | S2-9T15 | S4-9T15 |
Gel verification of hEF1a:CD79B-pTet:mRFP-Gal4VP16 SDM
we got no bands on the gel. however, when we talked to nicholas (who has experience with SDM), he said that bands may or may not appear on a gel depending on how much amplification happened. So we're going to run the SDM again and transform the PCR product directly without running it on a gel.