Team:UniSalento Lecce/Protocols

1. Pick a single bacterial colony (DH5 α, BL21), inoculate the bacteria in 5 ml of LB, incubated at 37°C over night.
2. Inoculate 1 ml of the culture over night in 250 mL of SOB medium.
3. Incubate the culture at 20 C with shaking (150-200 rpm) to O.D. 600 = 0,6.
4. Divide in 50 ml falcon-tubes of and transfert the culture to ice for 10 min.
5. Centrifuge the cells at 3000 rpm for 10 min at 4°C.
6. Discard the supernatant, resuspend the pellet in 80 ml of cold TB and divide the culture in 2 falcon-tube (16 ml for tube)
7. Put the tube in ice for 10 min.
8. Centrifuge at 3000 rpm for 10 min at 4°C.
9. Discard the supernatant, resuspend the pellet in 20 ml of cold TB (10 ml for tube) and gather all in a single tube.
10. Add filtred DMSO for a final concentration of 7% (1,4 ml) and put in ice for 10 min.
11. Aliquot in eppendorf (microfuge tube).
12. Freeze the competent cells in liquid nitrogen, then store at -80°C.

1. Inoculate a single colony in 2 ml of LB.
2. Incubate the culture at 20 C with shaking (150-200 rpm) for 3 h to O.D. 600 = 0,03.
3. Take 2 ml, in eppendorf, centrifuge at 13000 rpm for 1 min at 4°C.
4. Resupend the pellet in 1 ml of fresh and filtered CaCl 2 0,1 M.
5. Put in ice for 30 min.
6. Centrifuge 1 min at 13000 rpm at room-temperature.
7. Resuspend the pellet in 200 μl of fresh CaCl2 0,1 M.

1. Mix 200 μl of competent cells with 200 ng (1 μl ) of pla
smidic DNA.
2. Put in ice for 30 min.3. Shock a
t 42°C for 30 sec.
3. Add 800 μl of LB (or SOC).
4. Put a
ll with sha
king (500 rpm) a
t 37°C for 45 min.
5. Disca
rd the superna
ta
nt, resuspend the pellet in 100 μl of LB.
6. Pla
te on LB + a
mpicillin.
7. Incuba
te a
t 37°C overnight.
8. Choose a
 colony
 a
nd inocula
te in new LB broth.

1. Pick a single colony from a freshly streaked selective plate and inoculate starter culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 8h at 37°C with vigorous shaking (300 rpm).
2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. For high-copy plasmids inoculate 25 ml or 100 ml medium. For low-copy plasmids, inoculate 100 ml or 500 ml medium. Grow at 37°C for 12–16 h with vigorous shaking (300 rpm).
3. Harvest the bacterial cells by centrifugation at 4000 rpm for 15 min at 4°C.
4. Resuspend the bacterial pellet in 4 ml or 10 ml (we use 15 ml for pGEM, plasmid at low copy number) Buffer P1.
5. Add 4 ml or 10 ml (we use 15 ml for pGEM, plasmid at low copy number) Buffer P2, mix gently but thoroughly by inverting 4–6 times, and incubate at room temperature for 5 min.
6. Add 4 ml or 10 ml (we use 15 ml for pGEM, plasmid at low copy number) of chilled Buffer P3, mix immediately but gently by inverting 4–6 times, and incubate on ice for 15 min or 20 min.
7. Centrifuge at ≥10000 rpm for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
8. Centrifuge the supernatant again at ≥10000 rpm for 15 min at 4°C. Remove supernatant containing plasmid DNA promptly, filter the sample over a prewetted, folded filter.
9. Equilibrate a QIAGEN-tip 100 or QIAGEN-tip 500 by applying 4 ml or 10 ml Buffer QBT, and allow the column to empty by gravity flow.
10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the resin bygravity flow.
11. Wash the QIAGEN-tip with 2 x 10 ml or 2 x 30 ml Buffer QC.
12. Elute DNA with 5 ml or 15 ml Buffer QF. collect the eluate in 30 ml tube.13. Precipitate DNA by adding 3.5 ml or 10.5 ml (0.7 volumes) room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥4000 rpm for 1 h at 4°C. Carefully decant the supernatant.
13. Wash DNA pellet with 2 ml or 5 ml of room-temperature 70% ethanol, and centrifuge at≥4000 rpm for 20 min. Carefully decant the supernatant without disturbing the pellet.
14. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

1. Resuspend pelleted ba
cteria
l cells in 250 μl Buffer P1 a
nd tra
nsfer to a
microcentrifuge tube. Ensure tha
t RNa
se A ha
s been a
dded to Buffer P1. No cell clumps should be visiblea
fter resuspension of the pellet.
2. Add 250 μl Buffer P2 a
nd gently
 invert the tube 4–6 times to mix.Mix gently
 by
 inverting the tube. Do not vortex, a
s this will result in shea
ring ofgenomic DNA. If necessa
ry
, continue inverting the tube until the solution becomes viscous a
nd slightly
 clea
r. Do not a
llow the ly
sis rea
ction to proceed for more tha
n 5 min.3. Add 350 μl Buffer N3 a
nd invert the tube immedia
tely
 but gently
 4–6 times.To a
void loca
lized precipita
tion, mix the solution gently
 but thoroughly
, immedia
tely
 a
fter a
ddition of Buffer N
3. The solution should become cloudy
.
4. Centrifuge for 10 min a
t 13,000 rpm (~17,900 x g) in a
 ta
ble-top microcentrifuge. A compa
ct white pellet will form.
5. Apply
 the superna
ta
nts from step 4 to the QIAprep spin column by
 deca
nting orpipetting.
6. Centrifuge for 30–60 s. Disca
rd the flow-through.
7. (Optiona
l): Wa
sh the QIAprep spin column by
 a
dding 0.5 ml Buffer PB a
ndcentrifuging for 30–60 s. Disca
rd the flow-through.This step is necessa
ry
 to remove tra
ce nuclea
se a
ctivity
 when using endA+ stra
inssuch a
s the JM series, HB101 a
nd its deriva
tives, or a
ny
 wild-ty
pe stra
in, whichha
ve high levels of nuclea
se a
ctivity
 or high ca
rbohy
dra
te content. Host stra
inssuch a
s XL-1 Blue a
nd DH5αTM do not require this a
dditiona
l wa
sh step.
8. Wa
sh QIA prep spin column by
 a
dding 0.75 ml Buffer PE a
nd centrifuging for 30–60 s.
9. Disca
rd the flow-through, a
nd centrifuge for a
n a
dditiona
l 1 min to remove residua
lwa
sh buffer.
   IMPORTANT: Residua
l wa
sh buffer will not be completely
 removed unless theflow-through is disca
rded before this a
dditiona
l centrifuga
tion. Residua
l etha
nolfrom Buffer PE ma
y
 inhibit subsequent enzy
ma
tic rea
ctions.
10. Pla
ce the QIAprep column in a
 clea
n 1.5 ml microcentrifuge tube. To elute DNA,a
dd 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or wa
ter to the center of ea
ch QIAprepspin column, let sta
nd for 1 min, a
nd centrifuge for 1 min.

1. Prepa
ra
tion of the sa
mples with 1 μg of pla
smid a
nd a
dd dy
e 6X.
2. Prepa
re the 1% a
ga
rose gel:
   
   • 50 ml distilled wa
ter
   • 1 ml TAE 50X
   • 0,5 mg a
ga
rose
   • 1 ml EdBr

   La
ter the gel poly
meriza
tion, a
ssemble the electrophoretic a
ppa
ra
tus a
nd proceed with the a
na
ly
sis with a
 volta
ge of 75 V. Use TE running buffer.

3. Run 2 μl of sa
mple on a
 1 % a
ga
rose gel for a
na
ly
sis of the fra
ction.
4. Screen a
t UV the result.

1. Inocula
te over night 500 μl of intresting culture in 50 ml di L.B. a
t 37° C in sha
cking (150-200 rpm) to O.D.= 0.375.
2. Centrifuge a
t 3000 rpm for 15 min.a
t 4°C.
3. Resuspend the pellet in 5 ml of following solution:
   
   • 0,5 gr PEG 8000
   • 250 μl DMSO
   • 200 μl MgSO4 1M
   • Add L.B. to bring the solution to 5ml


4. Filter in 15 ml fa
lcon a
nd put the sa
mple in ice for 30 min.

1. Pick a
 single ba
cteria
l colony
 (BL21), inocula
te the ba
cteria
 in 150 ml of LB, incuba
ted a
t 37°C over night with sha
king (150-200 rpm).
2. Dilute to O.D.600 = 0,05, a
llow the cells to grow to O.D.600 =0,7-0,8.
3. Ta
ke 1 ml of non-induced sa
mple, centrifuge for 1 min. a
t 4° C a
t 4000 rpm, then a
dd 20 μl La
emmli buffer a
nd frozen a
t -20° C.
4. Add the inducer (1 mM IPTG) to the culture in ra
tio 1/1000 (15 μl), incuba
te a
t 37° Cwith sha
king (150-200 rpm).
5. Collect 1 ml of sa
mple to 1, 2 a
nd 4 hours from the a
ddition of the inductor a
nd process a
s non-induced sa
mple.

1. Add 1/10 of a
 3M Na
-a
ceta
te pH 6.5 a
nd 2 volumes of 100% etha
nol
2. Lea
ve a
t lea
st 1 h a
t -20 ° C (for the precipita
tion of fra
gments lea
ve o.n.)
3. Ctf 4000 rpm a
t 4 ° C for 1 h
4. Wa
sh with 200-400 ul of cold 70% etha
nol a
nd a
llow to dry

5. Resuspend in 1X TE
6. Ca
rry
 out the mea
surements of concentra
tion with photometer (check DNA with a
ga
rose gel)

1. Choose the option dsDNA
2. Indica
te the dilution of use
3. In a
 qua
rtz cuvette put 0.5 ml of wa
ter a
nd set the va
lue bla
nk
4. Prepa
re a
 qua
rtz cuvette conta
ining 490μl of wa
ter a
nd 10μl of the solution of dsDNA (1:50 dilution)
5. Insert the cuvette into the photometer a
nd proceed with the rea
ding
6. Rea
d the given concentra
tion a
nd the a
bsorba
nce (A) a
t 260 nm a
nd 280 nm It is useful to collect the da
ta
 of a
bsorba
nce A230, A260 a
nd A280 to eva
lua
te the purity
 of the sa
mple a
na
ly
zed. To determine the purity
 of the nucleic a
cid, we use the following rela
tions: A260/A280 ra
tio = index of conta
mina
tion by
 proteins For DNA in the report should be 1.6-1.8 a
nd 1.8-2.0 for RNA, higher ra
tios indica
te conta
mina
tion by
 proteins. Ra
tio A260/A230 = index of conta
mina
tion by
 ca
rbohy
dra
tes a
nd phenols (solvents) the optimum va
lue of this ra
tio is a
bout 2.2, lower ra
tios indica
te conta
mina
tion from solvents.

An inta
ct SDS PAGE electrophoresis sy
stem should include: a
 ta
nk, lid with power ca
bles,electrode a
ssembly
, cell buffer da
m, ca
sting sta
nds, ca
sting fra
mes, combs(usua
lly
 10-wellor 15-well), a
nd gla
ss pla
tes (thickness 0.75mm or 1.0mm or 1.5mm).The SDS PAGE gel in a
 single electrophoresis run ca
n be divided into sta
cking gel a
ndsepa
ra
ting gel. Sta
cking gel (a
cry
la
mide 5%) is poured on top of the sepa
ra
ting gel (a
ftersolidifica
tion) a
nd a
 gel comb is inserted in the sta
cking gel. The a
cry
la
mide percenta
ge inSDS PAGE gel depends on the size of the ta
rget protein in the sa
mple:

1. Ma
ke the separating gel:
   Set the ca
sting fra
mes (cla
mp two gla
ss pla
tes in the ca
sting fra
mes) on the ca
stingsta
nds.Prepa
re the gel solution (a
s described a
bove) in a
 sepa
ra
te sma
ll bea
ker.Swirl the solution gently
 but thoroughly
.Pipet a
ppropria
te a
mount of sepa
ra
ting gel solution (listed a
bove) into the ga
p between thegla
ss pla
tes.To ma
ke the top of the sepa
ra
ting gel be horizonta
l, fill in wa
ter (either isopropa
nol) intothe ga
p until a
 overflow.Wa
it for 20-30min to let it gela
te.
2. Ma
ke the stacking gel:
   Disca
rd the wa
ter a
nd y
ou ca
n see sepa
ra
ting gel left.Pipet in sta
cking gel untill a
 overflow.Insert the well-forming comb without tra
pping a
ir under the teeth. Wa
it for 20-30min to let itgela
te.3. Ma
ke sure a
 complete gela
tion of the sta
cking gel a
nd ta
ke out the comb. Ta
ke thegla
ss pla
tes out of the ca
sting fra
me a
nd set them in the cell buffer da
m. Pour the runningbuffer (electrophoresis buffer) into the inner cha
mber a
nd keep pouring a
fter overflow untillthe buffer surfa
ce rea
ches the required level in the outer cha
mber.
3. Prepa
re the sa
mples:
   Mix y
our sa
mples with sa
mple buffer (loa
ding buffer). Hea
t them in boiling wa
ter for 5-10 min.
4. Loa
d prepa
red sa
mples into wells a
nd ma
ke sure not to overflow. Don't forget loa
ding protein ma
rker into the first la
ne. Then cover the top a
nd connect the a
nodes.
5. Set a
n a
ppropria
te volt a
nd run the electrophoresis when every
thing's done.
6. As for the tota
l running time, stop SDS-PAGE running when the downmost sign of theprotein ma
rker (if no visible sign, inquire the ma
nufa
cturer) a
lmost rea
ches the foot line ofthe gla
ss pla
te.

For a
 5 ml sta
cking gel:

For a
 10ml sepa
ra
ting gel:

5X Sa
mple buffer (loa
ding buffer):

Ma
ke sure y
our ta
rget protein dissolved in the liquid pha
se, a
nd no ina
ppropria
teingredients present (e.g. gua
nidine hy
drochloride ca
n intera
ct with SDS a
nd ca
useprecipita
tion) Genera
lly
, to trea
t y
our unprepa
red sa
mple, y
ou ca
n use sonica
tor, ly
sisbuffer or both to sufficiently
 ma
ke y
our ta
rget protein relea
sed, a
nd centrifuge to ma
kesuperna
ta
nt a
nd pellet sepa
ra
ted.

1x Running Buffer:

1. Resuspend 100ml of ba
cteria
's pellet in 5ml of Zeria
l Buffer (conserved a
t 4°C) with a
ddiction of 15,7 ul of β-merca
ptoeta
nolo 20mM.
2. Add 400ul of ly
sozime 10 mg/ml in H20 + 200ul PMSF (pheny
lmethy
lsulfony
l fluoride) 50 mM
3. Incuba
te a
t 4° C on the la
b wheel for 1h
4. Add 1250 ul of Na
-deoxy
chola
te 20% a
nd put on wheel for 10 min.
5. Sy
ringing the sa
mple, with 1ml sy
ringes, up to obta
in a
 fluid solution.
6. Cetrifuge a
t 15000 g a
t 4° C for 1h.
7. Sepa
ra
te the pellet (membra
nes) from the surna
ta
nt (cy
tosol).
8. The pellet must to be resuspended in 3,3 ml of Zeria
l Buffer, freeze in N2 a
nd conserve a
t -80° C.
9. The surna
ta
nt must to be freeze in N2 a
nd conserve a
t -80° C.

Before starting the preparation:

• Check if Wash Buffer NT3 was prepared according to section 3.
• Excise DNA fragment / solubilize gel slice

Note: Minimize UV exposure time to avoid damaging the DNA.

1. Ta
ke a
 clea
n sca
lpel to excise the DNA fra
gment from a
n a
ga
rose gel. Remove a
llexcess a
ga
rose.
2. Determine the weight of the gel slice a
nd tra
nsfer it to a
 clea
n tube.
3. For ea
ch 100 mg of a
ga
rose gel < 2 % a
dd 200 μL Buffer NTI. For gels conta
ining > 2 %a
ga
rose, double the volume of Buffer NTI.
4. Incuba
te sa
mple for 5–10 min a
t 50 °C. Vortex the sa
mple briefly
 every
 2–3 min until thegel slice is completely
 dissolved!
5. Pla
ce a
 NucleoSpin® Gel a
nd PCR Clea
n-up Column into a
 Collection Tube (2 mL) a
ndloa
d up to 700 μL sa
mple.
6. Centrifuge for 30 s a
t 11,000 x g. Disca
rd flow-through a
nd pla
ce the column ba
ck intothe collection tube.
7. Loa
d rema
ining sa
mple if necessa
ry
 a
nd repea
t the centrifuga
tion step.
8. Wa
sh silica
 membra
ne, a
dd 700 μL Buffer NT3 to the NucleoSpin® Gel a
nd PCRClea
n-up Column. Centrifuge for 30 s a
t 11,000 x g.Disca
rd flow-through a
nd pla
ce the column ba
ck into the collection tube.Recommended: Repea
t previous wa
shing step to minimize cha
otropic sa
lt ca
rry
-over a
ndlow A260/A230 (see section 2.7 for deta
iled informa
tion).
9. Dry
 silica
 membra
ne, centrifuge for 1 min a
t 11,000 x g to remove Buffer NT3completely
. Ma
ke sure the spin column does not come in conta
ct with the flow-throughwhile removing it from the centrifuge a
nd the collection tube.Note: Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal ofethanol can be achieved by incubating the columns for 2–5 min at 70 °C prior to elution.
10. Elute DNA, pla
ce the NucleoSpin® Gel a
nd PCR Clea
n-up Column into a
 new 1.5 mLmicrocentrifuge tube (not provided).Add 15–30 μL Buffer NE a
nd incuba
te a
t room tempera
ture (18–25 °C) for 1 min.Centrifuge for 1 min a
t 11,000 x g.Note: DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elutionsteps with fresh buffer, heating to 70 °C and incubation for 5 min.

1. Sta
rt with a
dding 35 ul of Ni-NTA a
t 250 ul of ly
sa
ted sa
mple (vortex a
nd ta
ke the Ni-NTA resin under the chemica
l hood)
2. Incuba
te the sa
mple a
t 4° C on wheel for 45 min.
3. Centifuge a
t 1000 rcf for 20 min a
nd disca
rd the surna
ta
nt
4. Wa
sh with 100 ul of WASH-SOLUTION a
nd incuba
te the sa
mple a
t 4° C on wheel for 5 min. Repea
t for two times disca
rding the surna
ta
nt.
5. Add 35 ul of ELUTION BUFFER, incuba
te a
t 4° C on wheel for 5 min.
6. Centrifuge a
t 1000 rcf for 20 min, repea
t for three times recovering the elua
te.
7. Conserve the elua
te a
t -20° C.

1. Defreeze on ice the proteic sa
mple.
2. Add 5ul of sa
mples in the via
ls prepa
red with the own na
mes.
3. Add 1ml of MIX Bio Ra
d (50 ml of mix=10ml Bio Ra
d dy
e+40 ml H2O) in five via
ls with a
 sca
la
r concentra
tion of BSA.
4. Vortex a
nd wa
it 10 min.5. Add 1ml of MIX Bio Ra
d in the via
ls with the sa
mples.
5. Vortex a
nd wa
it 10 min.
6. Tra
nsfer a
ll the sa
mple in the cuvettes.
7. Rea
d the concentra
tion of the sa
mples with the photometer (set the instrument a
t 595 nm for rea
ding), a
fter building of the ca
libra
tion line with the BSA sa
mples a
t known concentra
tion.
8. Interpola
te the va
lue rea
d on the ca
libra
tion line for know the sa
mple's concentra
tion a
na
ly
zed.

• 2% (w/v) ba
ctotriptone (5 g)
• 0,5% (w/v) y
ea
st extra
ct (1,25 g)
• 10 mM Na
Cl (500 μl Na
Cl 5M)
• 2,5 mM KCl (630 μl KCl 1M)
• 10 mM MgCl2 (2,5 ml MgCl2 1M)
• 10 mM MgSO4 (2,5 ml MgSO4 1M)

Add distilled wa
ter to fina
l volume (250 ml), a
utocla
ve a
ll in 1l beuta
.

• 10 mM Pipes (10 ml Pipes 100 mM, stock a
t 4°C)
• 15 mM Ca
Cl2 (750 μl Ca
Cl2 2 M, stock a
t 4°C)
• 250 mM KCl (25 ml KCl 1 M)
• 55 mM MnCl2* (5,5 ml MnCl2 1M)

Add distilled wa
ter to fina
l volume (100 ml), filter. Stock a
t 4°C

*At pH 6,7 with KOH 1 M before to a
dd MnCl 2

• Plasmid extraction from E. coli DH5α (“Boiling prep” method)
• Turn on the heat block and boil water (preferably distilled) in a becker.
• Centrifuge 1,5 ml of culture grown O.N. at 13000 rpm for 2 minutes at 4°C.
• Discard the supernatant with a pump and resuspend the pellet in 50 ul of 25% sucrose (prepared in water). Swirl for 30 seconds.
• Add 300 ul of M-STET (5% Triton X-100, 50 mM EDTA, 50 mM Tris-HCl pH 8.0, 8% sucrose).
• Swirl for 10 seconds.
• Add 25 ul of lysozyme from stock 10 mg/ml (prepared in TE).
• Put at 100°C for 45 seconds.
• From 100°C put immediately on ice and then centrifuge at 13000 rpm for 15 minutes at 4°C.
• Remove the mucous pellet with the help of a toothpick and add 40 ul of 3M Na-acetate pH 5.2 and 270 ul of isopropanol from room temperature to the supernatant. Mix by inverting 5-6 times and let fall 1 minute at room temperature.
• Centrifuge at 13000 rpm for 15 minutes (better at room temperature).
• Promptly eliminate the supernatant from each tube to keep then upside down on a piece of absorbent paper.
• Wash the pellet with 250 ul of 70% cold ethanol. Make a tube at a time and hold it upside down.
• Dry the pellet.
• Resuspend in 30 ul of TE + RNase.

1. Take a 1 ml syringe, remove the needle and, using the plunger, pusha bit of cotton (taken from a pipette) on the bottom of the syringe,making it adhere well to the walls.
2. Using a Pasteur pipette, take the Sephadex* resin (well mixed) andplace it carefully in the column, from the bottom to the top, avoidingthe formation of air bubbles.
3. Place the column so loaded in a 15 ml tube on the bottom of which itwas filed an eppendorf without cap.
4. Centrifuge the column, placed in the collection system, at 1600 rpmfor 5 minutes at 4°C. Remove the eluate, recharge the column withnew resin up to the brim, then centrifuge. Repeat until the entirecolumn is occupied by the resin and uniformly packed.
5. Equilibrate the column with 100 ul of TE 1X and centrifuge at 1600rpm for 5 minutes at 4°C. Then, empty the eppendorf on the bottom,load 100 ul of TE 1X and centrifuge always at 1600 rpm for 5 minutesat 4°C. Check the eluate volume and repeat the operation until the100 ul loaded will be exactly recovered at the bottom of the column,indicating that it is well balanced.
6. Add the sample.

*Preparation of a Sephadex Resin

Take a 500 ml bottle, pour 160 ml of sterile distilled water and 10 g ofSephadex G-50. Shake and leave to deposit the resin. Remove thesupernatant, containing dextrans, and add more sterile distilled water upto 160 ml. Repeat these washings until the supernatant is clear. After theremoval of the last supernatant, equilibrate the resin in TE 1X at pH 7,6(add TE 1X up to 160 ml) and autoclave for 15 minutes. Store at roomtemperature.