dspB Track
- Plate taken out at 11:15pm
- no colonies
- seems like the ligation had failed; therefore, re-do ligation between his dspB and ccdB chlor backbone
Ligation
- Protocol: "Ligation" protocol from Biobrick
- 5uL vector: 1.5uL his
- Tube: his + ccdB -> HC
- Incubate at room temperature (RT) for 20 minutes
Transformation of HC
- Protocol: SOP; used competent cell #2
- Changes: use 5uL instead of 1uL of ligation mix
- Plates in 37C incubator at 3:05pm
Rafael's Method
PNK Treatment of DNA Ends on primers
- Primers: dspB fw rxn1, dspB rev rxn1, dspB fw rxn2, dspB rev rxn2
REAGENTS | 1 RXN VOLUME (uL) |
PNK Stock | 1 | |
T4 ligase buffer | 1 |
Primer | 8 |
- Tubes: dspB fw rxn1 PNK, dspB rev rxn1 PNK, dspB fw rxn2 PNK, dspB rev rxn2 PNK
PCR using Rafael's method
PCR Master mix
Reagent | 1x rxn volume (uL) |
5x rxn buffer | 5 |
10mM dNTP | 1 |
sdH2O | 6.65 |
Phusion polymerase | 0.1 |
MgCl2 | 2 |
DMSO - 5% | 1.25 |
gDNA | 3 |
Total | 25 |
- To PCR tubes, add 3uL of rev + fw to each respective tube
- fw+rev rxn 1 -> tube 1
- fw+rev rxn 2 -> tube 2
Tubes: rxn1, rxn2
PCR Cycles:
- 98C @ 2min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 4:08pm
End: 5:10pm
Restriction Digest
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL) |
Buffer 2 | 2 | |
BSA | 0.2 |
DNA | 10 |
ddH20 | 5.8 |
Total | 20 |
Enzymes: Use XbaI and PstI (1uL each)
DNA: ccdb-chlor backbone
Digest for 2 hours
Assembling the final construct
Restriction Digest
Restriction Digest Supermix
REAGENTS | 1 RXN VOLUME (uL) |
Buffer 2 | 2 | |
BSA | 0.2 |
DNA | 10 |
ddH20 | 5.8 |
Total | 20 |
Enzymes: Use EcoRI and SpeI (1uL each)
DNA: prefix and dspB NH
Digest for 2 hours
Gel verification on RD, ccdB Chlor, PCR products
- Protocol: biobrick "digest" protocol
- Changes: load 20uL into each well; 10uL for ladder
Gel orientation:
Gel orientation
1 kb ladder | pre | dspB | 100bp ladder | 1 | 2 | ccdB XP |
Machine conditions: 0.5x TBE buffer, 100V, 60min
Results:
- Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)
ccdB XP gel weight: 0.1161g
pre gel weight: 0.1218g
Resuspended in 40uL of buffer
Nanodrop:
- [ccdB XP] = 4.6ng/uL
- [pre] = 7.0ng/uL
PCR using Rafael's method Attempt #2
PCR Master mix
Reagent | 1x rxn volume (uL) |
5x rxn buffer | 5 |
10mM dNTP | 1 |
sdH2O | 6.65 |
Phusion polymerase | 0.1 |
MgCl2 | 2 |
DMSO - 5% | 1.25 |
gDNA | 3 |
Total | 25 |
- To PCR tubes, add 3uL of rev + fw to each respective tube
- fw+rev rxn 1 -> tube 1
- fw+rev rxn 2 -> tube 2
Tubes: rxn1, rxn2
PCR Cycles:
- 98C @ 2min
- Cycle 27x:
- 98C @ 10 sec
- 72C @ 30 sec
- 72C @ 40 sec
- 72C @ 10 min
- 10C @ hold
Start: 9:55pm
End: 10:57pm
Gel verification PCR products Attempt #2
- Protocol: biobrick "digest" protocol
Gel orientation:
Gel orientation
Quick load 100 bp ladder(last year) | 2b | W | Quick load 100bp ladder (this year) | 1 | 2 | ccdB XP |
Machine conditions: 0.5x TBE buffer, 100V, 45min
Results:
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