Objective
- Run DNA gel with QC product
DpnI digest of product (and transformation, if applicable)
Re-transform pTXB1 dd + insert samples with greater [DNA]Replate transformations from yesterday (no colonies this morning)- Redo QC with pTXB1 control
- Run gel with serial dilutions of pKK223-3 and another with both cleaned and non-cleaned BamHI-digested pKK223-3
Bench work
- DNA gel with QC product from yesterday
- Lane 1 Ladder
- Lane 2 pMXB10 @ 10ng/μL (shouldn't actually show up)
- Lane 3 pMXB10 QC +His
- Lane 4 pMXB10 QC (-) control
- Lane 5 ----
- Lane 6 ~600ng of pKK223-3 miniprep 7/26 by Kay Wang
- Lane 7 ~300 ng of same sample
- Lane 8 ~150 ng
- Lane 9 ~75 ng
- Lane 10 ~32.5 ng
- Lane 11 ~16.25 ng
- BamHI digest of pKK223-3 miniprep 7/26 by Kay Wang
- Two samples, identical: 20μL 128ng/μL pKK223-3, 5μL NEB3 10X 5μL BamHI, 0.5μL BSA 100X, 19.5μL dH2O. 37°C for 2 hours.
- Freeze one digest and clean-up the other with Promega Wizard® DNA clean up kit
- DNA gel #2 for the day:
- Lane 1 Ladder
- Lane 2 ~256ng of BamHI unclean digest of pKK223-3 above
- Lane 3 ~128ng of same sample
- Lane 4 ~64ng
- Lane 5 ~32ng
- Lane 6 ~16ng
- Lane 7 ~8ng
- Lane 8 ----
- Lane 9 ~256ng of BamHI cleaned digest of pKK223-3 above
- Lane 10 ~128ng of same sample
- Lane 11 ~64ng
- Lane 12 ~32ng
- Lane 13 ~16ng
- Lane 14 ~8ng
- QuikChange 2 stage with pMXB10 and +His primers and pTXB1 as (+) control
Results
- Kathryn Muratore 13:44, 10 August 2011 (EDT): This shows an incomplete digest by BamHI. In Gel #1, you see that the uncut plasmid runs at ~6500 and ~3000 bp. Cut plasmid is expected to run as two bands at 4516 and 268 bp.
- Top band is uncut plasmid (relaxed)
- 2nd band is plasmid cut once (4584 bp)
- 3rd band is plasmid cut twice (4516 bp)
- 4th band is uncut plasmid (supercoiled)
- 268 bp piece of DNA is not visible at these load levels and/or was run off the gel.
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AU CHEM-570 Lab Prep
