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Team:Peking/Notebook/MChen
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Mei Chen's Notes
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July
July
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7.1-7.7
I wanted to get CrtE, CrtB, CrtI, CrtY, CrtZ, VioA, VioB, VioC, VioD and VioE for producing CrtEBI, CrtEBIY, CrtEBIYZ, VioABCE, VioABDE and VioABCDE.
1. Extract DNA from the registry 2. Add 10μl ddH2O, leave the water in the well for 30 sec(red).
BBa_K118014 rbs+crtE 2-18H/2010 pSB1A2
BBa_K118013 rbs+crtY 2-18F/2010 pSB1A2
BBa_K118006 rbs+crtB 2-16N/2010 pSB1A2
BBa_K118005 rbs+crtI 2-16L/2010 pSB1A2
BBa_K274003 VioABDE 3-20H/2010 pSB1K3
BBa_K274004 VioABCE 3-20J/2010 pSB1K3
2. Pipette 2μl of the resuspended DNA transform into the Trans 5a competent cells.
Each tube: Competent cells 50μl + DNA 2μl
3. Picking a single colony and inoculate for 15 hours(in 5ml antibiotics culture)
4. Miniprep and use spectrophotometer to estimate the concentration of DNA(50×dilution)
VioABDE A260=0.027 conc=1.3401μg/ml
VioABCE A260=0.022 conc=1.0782μg/ml
CrtY A260=0.047 conc=2.3349μg/ml
CrtI A260=0.078 conc=3.8963μg/ml
CrtZ A260=0.044 conc=2.2146μg/ml
CrtE A260=0.087 conc=4.3482μg/ml
CrtB A260=0.055 conc=2.7299μg/ml
5. PCR for VioA, VioB, VioC, VioD, VioE
The primer for VioA, VioB, VioC, VioD and VioE are:
ViolaA_For ccggaattcgcggccgcttctagATGAAACATTCTTCCGATATCTGCATTGTTGG ViolaA_Rev aaactgcagcggccgctactagtaTCACGCGGCGATACGCTGCA ViolaB_For ccggaattcgcggccgcttctagATGAGCATTCTGGATTTCCCGCGTATC ViolaB_Rev aaactgcagcggccgctactagtaTTAGGCCTCGCGGCTCAGTTTG ViolaC_For ccggaattcgcggccgcttctagATGAAACGTGCGATTATCGTTGG ViolaC_Rev aaactgcagcggccgctactagtaTCAATTCACGCGACCAATCTTG ViolaD_For ccggaattcgcggccgcttctagATGAAGATTCTGGTCATTGGTG ViolaD_Rev aaactgcagcggccgctactagtaTCAGCGCTGCAAAGCATAAC
ViolaE_For ccg gaattc gcggccgct tctagATGGAGAACCGTGAGCCAC
ViolaE_Rev aaa ctgcag cggccgct actagtaTTAGCGCTTGGCCGCGAAA
dd 11.7μl
5×phusion HF buffer 4μl
2.5MdNTP 1.6μl
For 1μl
Rev 1μl
Template 0.5μl
(chill on ice)
Phusion pol 0.2μl
I did the PCR three times, VioB, VioC and VioE hadn’t result. VioA and VioD were extracted from the gel.
7.8-7.14
1. Double digest for CrtE、CrtB、CrtI、CrtY、CrtZ
CrtB、CrtY、CrtZ: EcoRI and XbaI
CrtE、CrtI: EcoRI and SpeI
NEB:
CrtE or CrtI 10μl
10×EcoRI buffer 2μl
EcoRI 1μl
SpeI 1μl
ddH2O 6μl
Total 20μl
Takara:
CrtB or CrtY or CrtZ 10μl
10×buffer(1×M) 2μl
EcoRI 1μl
XbaI 1μl
ddH2O 6μl
Total 20μl
After double digestion, electrophoresis in 1.5% agarose gel to test the results and the result of CrtI was wrong. I did the double digestion for CrtI again and the result was wrong again.
2. Ligate CrtE and CrtB
CrtB 2μl
CrtE 6μl
10×T4 buffer 1μl
T4 DNA Ligase 1μl
3. Transform CrtEB, pick five single colonies and inoculate(in 5ml antibiotics culture), miniprep by Trans kit, identified by XbaI and PstI digestion. 4.
NEB:
CrtEB 5μl
10×3 buffer 2μl
XbaI 1μl
PstI 1μl
ddH2O 11μl
Total 20μl
5. Sequence by Hua Da company. Two results were right 6. 7. Transform BBa_K118005 rbs+crtI and double digestion again, but the result was still wrong. The idea for producing CrtEBI, CrtEBIY and CrtEBIYZ by myself was given up because of the time limition. 8. 9. Gradient PCR for VioB and VioE 10.
Easymix 10μl
ddH2O 8.5μl
Primer For 0.5μl
Primer Rev 0.5μl
Template 0.5μl
Total 20μl
Gradient:
5 50.4℃
6 53.0℃
7 55.8℃
8 58.5℃
9 61.0℃
Electrophoresis in 1.5% agarose gel to test the results of PCR, VioE had results.
6. PCR for VioB by taq polymerase
TransEasymix 10μl
ddH2O 8.5μl
Primer For 0.5μl
Primer Rev 0.5μl
Template 0.5μl(VioABDE)
Total 20μl
Gradient:
5 52.5℃
6 55.1℃
7 57.8℃
8 60.5℃
9 63.0℃
Electrophoresis in 1.5% agarose gel to test the results of PCR VioB, five gradients had results. All were extracted from the gel and sequence, gradient 6 and gradient 7 had no mutation.
7.15-7.22
1. Transform VioABCDE, double digestion VioABCDE for VioC(BamHI, BglII)
Takara:
VioABCDE 10μl
BamHI 1.5μl
BglII 1.5μl
10×K buffer 2μl
ddH2O 5μl
total 20μl
Control
VioABCDE 10μl
BamHI 2μl
10×K buffer 2μl
ddH2O 6μl
total 20μl
37℃ 4 hours
Or
37℃ overnight
Electrophoresis in 1.5% agarose gel, the result of BamHI digestion was the same as double digestion.
2. Gradient PCR for VioC
Easymix 10μl
ddH2O 8.5μl
Primer For 0.5μl
Primer Rev 0.5μl
Template 0.5μl(VioABCDE)
Total 20μl
Gradient
5 46.4℃
6 49℃
7 51.8℃
8 54.5℃
9 57℃
Electrophoresis in 1.5% agarose gel, no result.
I did the transformation, double digestion and PCR twice but the result was the same. The idea for producing VioABCE, VioABDE and VioABCDE by myself was given up because of the time limition.
7.23-8.17
I began to do the characterization for CrtEBI(K274100) and CrtEBIY(K274200).
CrtEBI(K274100) and CrtEBIY(K274200) were ligated to constitutive promoter (BBa_J23103) and terminator (B0015). All the protocols followed protocols above. Unfortunately, I failed in ligating the CrtEBIY and terminator (B0015) though I did this experiment many times in different conditions. The conditions included
1. ligase with different companies(NEB and Trans)
2. different ligation time(1, 2, 4, 12 hours)
3. different reaction systems
(1)
XP CrtEBIY(insert) 2μl 1μl 4μl
SP terminator(vector) 6μl 7μl 4μl
10×T4 buffer 1μl
T4 DNA Ligase 1μl
Total 10μl
(2)
XP CrtEBIY(insert) 4μl 2μl 8μl
SP terminator(vector) 12μl 14μl 8μl
10×T4 buffer 2μl
T4 DNA Ligase 2μl
Total 20μl
4. different temperatures(16℃ and 25℃).
The results were on our wiki, so I didn’t repeat here.
