August 5th, 2009
- Possible source of problem: Satoe and Chichi recommend larger reaction volume because glycerol from restriction enzyme stock can inhibit reaction
- larger reaction volume (30μL+) also recommended to have smaller template DNA concentration
- Concentration of DNA stock (made on 6/21/09) is 426 μg/mL or .46ng/μL
- Concentration of RFP: 5.56 μg/mL or 5.5 ng/μL
- Concentration of mCherry: 6.6 μg/ml or .46 ng/μL
Digest:
50μL reaction mCherry RFP Control
DNA 1 μL 5 μL 5 μL 1μL
(plasmid) (insert) (insert) (GFP-Kan)
NEB Buffer 4 1 μL 5 μL 5 μL 5μL
10x BSA 5 μL 5 μL 5 μL 5μL
Enzyme .25 + .25 .25 + .25 .25 + .25 .25μL PacI
ddH2O 38.5μL 34.5μL 34.5μL 39μL
Total 50μL 50μL 50μL 50μL
- Run for 3 hours
- Note: .25μL of enzyme should digest 2.5 μg of DNA, so we have a 5-fold excess of enzyme for plasmid, 50-fold for insert
- Gel here!
- Not much RFP/mcherry visible
- Gel Purification necessary?
- Digest possibilities:
- PacI/BglII -> Buffer 2 and BSA at 37°C, extra enzyme
- AscI/SpeI -> Buffer 4 and BSA at 37°C
- SpeI/BglII -> Buffer 2 and BSA at 37°C, extra enzyme
-Extra digest to double check SpeI cuts
- Control (no enzyme), buffer 2
Digest:
PacI/BglII AscI/SpeI SpeI/BglII Control
NEB Buffer 2, 1μL 4, 1μL 2, 1μL 2, 1μL
BSA 1 μL 1 μL 1 μL 1 μL
Enzyme .75 + .75 .5 + .5 .75 + .75 -
ddH2O 5.5μL 6 μL 5.5 μL 7 μL
DNA 1 μL 1 μL 1 μL 1 μL
Total: 10 μL reactions
- Ran restriction digest
- Gel inserted here
- 3 hour digest worked! Despite low volume and high enzyme concntration
- PacI and AscI both work (fragment size is correct)
Will stop AscI/PacI digest at 3 hours
- Also tried gel purification of mcherry/RFP
- Gel purification
- Added 600μL ADB buffer
- elute in 20μL elution buffer, incubate for 1 minute then spen down
- check on gel to confirm purity, use leftover digest material as control
- take concentration
20 μL reaction
DNA: 10 μL
Buffer 4: 2 μL
BSA: 2 μL
AscI/PacI: .5 + .5
ddH2O: 5μL
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