Team:Goettingen/NoteBook w-1

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

 

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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

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

 

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

 

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

 

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

 

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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

è Our PfuS (polymerase) activity is slow ca. 500 bp/min