Try to get the ligation and or transformation to work for the BSA-intein constructs.
- Gel purification
- Gel purify the double-digested vectors and insert from yesterday following the manufacturer's protocol
- 1.2% agarose gel, stained with EtBr
- taping together 4 lanes on 14-well comb allows 60μL to be loaded without overflow
- 50μL NheI/SapI/Phosphatased pTXB1His1 + 10μL loading buffer
- 1.2656g - 1.0282g = 237.4mg agarose → 240μL Membrane Binding Solution
- 10μL 1KB DNA ladder
- 50μL NheI/SapI BSA-PCR + 10μL loading buffer
- 1.4189g - 1.0299g = 389mg agarose → 390μL Membrane Binding Solution
- 50μL NheI/SapI/Phosphatased pTXB1 + 10μL loading buffer
- 1.2834g - 1.0295g = 253.9mg agarose → 260μL Membrane Binding Solution
- I incubated the agarose on the column for 1 minute this time (as stated in instructions)
- I centrifuged the washed columns "without the lid" using the microfuge and manually holding the "on" button (this is not very safe, but not dangerous if you are careful and others are not in the area.)
- → store @ -10°C
- use digested DNA from last week, not today's
- O/N 16°C ligation protocol
- include a negative control this time
- 5μL Insert + 10μL Vector(His) + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
- 5μL Insert + 10μL Vector + 2μL ligase buffer + 2μL H2O + 1μL T4 DNA Ligase
- 10μL Vector(His) + 2μL ligase buffer + 7μL H2O + 1μL T4 DNA Ligase
- 5μL Vector + 2μL ligase buffer + 12μL H2O + 1μL T4 DNA Ligase
- There was not enough digested pTXB1 to use 10μL in negative control
- → 16°C O/N
- Use PCR machine as cold block
- Re-plate transformation
- 100μL of yesterday's transformations on 2 separate LBAmp100 plates (one for each ligation)
- → 37°C O/N
Yesterday's plates both had of colonies. After some poking around, we realized that the Ampicillin stocks made on 6/20 are only 10 mg/mL not 100 mg/mL. The plates poured yesterday were the first batch using the new Amp and are thus LBAmp10 not LBAmp100. I will be re-plating another 100μL on new LBAmp100 plates today.
- Looks good. Equal volumes of insert and vector will be fine. [DNA] calculations are based on 8.5μL each.
- Kathryn Muratore 09:59, 28 July 2011 (EDT): I did not get rid of the background before quantifying this gel previously. A new sheet has been added for the background subtracted values. This leads to a modest decrease in [DNA] and an increase in I:V.