Team:Goettingen/NoteBook w-1
From 2013.igem.org
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Restriction digestion of pGP172 with SacI
Restriction digestion of pGP172 with SacI
Components |
Volume (L) |
1500 ng plasmid |
20 |
SacI |
4 |
Fast Digest® buffer |
4 |
HPLC H2O |
12 |
TOTAL |
40 |
Incubation at 37 oC for 2 hours
1 2 3 4 5 6 7 8 9 10 11 12 13 14 |
1: 100kb ladder 2: clone 2 undigested 3: clone 2 digested 4: clone 33 undigested 5: clone 33 digested 6: clone 36 undigested 7: clone 36 digested 8: clone 34 undigested 9: clone 34 digested 10: insert (ull length) 11: insert (DAC domain) 12: pGP172 undigested 13: pGP172 digested 14: 1kb ladder
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Transformation of DH5α E. coli strain
Transformation of DH5α E. coli strain
Materials needed:
Liquid LB medium
LB agar plates with ampicillin (100 g mL-1)
Procedure:
Put your liagtion samples on ice, defreeze 200 L of your competent E. coli cells on the top of the ice and add the cells to your ligation samples. Mix it carefully.
Incubate the eppendorf reaction tubes for 30 min on ice, transfer the tubes for 90 sec to 42 oC (heat shock) and put them back for 5 min on ice.
Add 800 L LB medium (without antibiotic) to the cells, and incubate the cells for 1 hour at 37 oC with agitation.
Add 100 L of the culture onto LB agar plate (containing antibiotic) and spread them using a spreader.
The remaining cells are collected by centrifugation for 1 min at 13,000 rpm and remove the supernatant leaving 100 L to re-dissolve the pellet.
Plate this concentrated cells as well (as a backup)
Incubate the cells overnight at 37 oC
Later, the plates should be stored at 4 oC
We did 6 tubes (so, 12 plates)
Tube Number |
Content |
1 |
pGP172+dacA (both SacI&II digested) |
2 |
pGP172+CD (both SacI&II digested) |
3 |
pGP172 only (SacI&II digested) with ligation |
4 |
Competent cells only |
5 |
pGP172 only (SacI&II digested) without ligation |
6 |
Undigested pGP172 |
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Restriction Digestion of SacI and SacII, ligation of dacA to pGB172
Restriction Digestion of SacI and SacII, ligation of dacA to pGB172
DacA (treated previously with SacI) now with SacII
PCR purification
Qiagen® kit method
pGP172 (treated with SacI and SacII), DacA (treated with SacII), CD (treated with SacII)
Removal of the 5’ phosphate group with alkaline phosphatase in the backbone to prevent self ligation
Components |
Volume (L) |
pGP172 (SacI&II) |
30 |
Alkaline phosphatase |
1 |
Fast Digest® buffer |
4 |
HPLC H2O |
5 |
TOTAL |
40 |
Incubation at 37 oC for 30 min
PCR purification (Qiagen® kit method)
1 % agarose gel
Measurement of concentration
Vector/insert |
Concentration (ng mL-1) |
dacA (SacI&II) |
12.4 |
CD (SacI&II) |
9.1 |
pGP172 (SacI&II) |
14.1 |
pGP172 (SacI&II) |
17.9 |
Ligation
Components |
Volume (μL) |
Components |
Volume (μL) |
25-50 ng pGP172 |
2.2 |
25-50 ng pGP172 |
2.2 |
5X dacA (SacI&II) |
12.1 |
5X CD (SacI&II) |
16.5 |
T4 ligase buffer |
2 |
T4 ligase buffer |
3 |
T4 DNA ligase |
2 |
T4 DNA ligase |
3 |
HPLC H2O |
1.7 |
HPLC H2O |
5.3 |
TOTAL |
20 |
TOTAL |
30 |
Components |
Volume (L) |
25-50 ng pGP172 |
2.2 |
T4 ligase buffer |
1 |
T4 DNA ligase |
1 |
HPLC H2O |
5.8 |
TOTAL |
10 |
Incubation at 16 oC for overnight
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PCR purification with Qiagen® kit method
PCR purification with Qiagen® kit method
PCR purification
Qiagen® kit method
pGP172 (treated previously with SacI)
pGP172 (treated previously with SacII)
DacA (full length) approx. 900 bp
CD (treated previously with SacI)
Restriction Digestion
pGP172 (treated previously with SacI) now with SacII
pGP172 (treated previously with SacII) now with SacI
DacA (full length) with SacI
CD (treated previously with SacI) now with SacII
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Repeat the PCR of dacA with different primer concerntrations
Repeat the PCR of dacA with different primer concerntrations
Re-running of PCR. This time with 4μL of 5μM forward and 4 μL of 5μM reverse primers. Also, the extension time changed to 2 min (instead of 1 min). Rest of the parameters are the same.
Components |
Volume (L) |
Forward primer (5 M) |
4 |
Reverse primer (5 M) |
4 |
dNTPs (12.5 M) |
2 |
L. monocytogenes complete DNA |
2 |
5X HF Buffer |
10 |
PfuS (polymerase) |
1 |
HPLC H2O |
27 |
TOTAL |
50 |
Gel picture at the end of the day
PCR purification of CD
Qiagen® kit method
We eluted the DNA from the column with 30 L of elution buffer (as per the protocol 50 L of the elution buffer is used)
Restriction Digestion of CD with SacI
Components |
Volume (L) |
CD (ca. 600 bp) |
20 |
Fast Digest® SacI |
4 |
Fast Digest® buffer |
4 |
HPLC H2O |
12 |
TOTAL |
40 |
Incubation at 37 oC for 2 hours
Verified the digestion with 1 % agarose gel
1 2 3 4 5 6 7 8 9 |
1: 100kb ladder 2: L.monocytogenes domain after digestion with SacI 3: pGP172 after digestion with SacI 4: pGP172 digested with SacI 5: l. monocytogenes PCR product 6: l. monocytogenes PCR product 7: positive control (800bp) 8: one Primer used 9: 1kb ladder
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è Our PfuS (polymerase) activity is slow ca. 500 bp/min