STANDARD OPERATING PROCEDURE (SOP)
TITLE
SOP #:
Written by
Approved by
Issued
Revised
Version
Number e.g. P-SOP-005-
Name 1
Name 1
Date first issued
Date of revision
000
Page 1 of 5
1.
INTRODUCTION
Short description of contents and aims:
e.g.
Standard procedure for cloning of PCR products using TOPO cloning kit from Invitrogen. Spesifically
written for Zero Blunt® TOPO® PCR Cloning Kit for Sequencing and TOPO TA Cloning® Kit for
Sequencing with TOP10 Chemically Competent cells (referred to as E.coli in the text).
This procedure can also be used for other TOPO cloning kits (vectors with blue/white selection gene)
from Invitrogen. The user then have to incorporate steps regarding the use of X-gal as a part of the
procedure. This is particularly important with regard to the risk assessment; the risk will be
significantly greater when using X-gal since it is diluted in formamide.
2.
SAFETY
General laboratory safety applies.
User must read bullet points 6. Procedures and 7. Risk assessment before starting the lab work. If the
risk assessment is not complete, please report to person responsible for the lab or safety
representative.
State special safety precautions that apply to this SOP;:
e.g.
When using other cloning kits (vectors with blue/white selection gene) which requires the use of Xgal, the X-gal must be applied to the plates under the fume hood. The user must have had necessary
training in the use of fume hoods, read I-SOP-001-Fumehood(1972) or other I-SOP.
Risk assessment conclution (select on of the boxes below):
Select one of the following risk assessment based on section 7 o these procedure.
Risk
assessment
There is a small/minimal overall risk associated with the use of this
procedure, provided that this procedure is followed.
(or)
Risk
Assessment
There is a MEDIUM overall risk associated with the use of this procedure,
provided that this procedure is followed. All user must act cautiously where
and when is required.
(or)
Risk
Assessment
There is a HIGH overall risk associated with the use of this procedure,
provided that this procedure is followed. All user must act cautiously where
and when is required or action to improve the procedure should be
considered.
Postal address: UiO, Department of Biosciences, P.O. Box 1066 Blindern, N-0316 Oslo
Version: 000
Number e.g. P-SOP-005-
Page 2 of 5
3.
NECESSARY SAFETY EQUIPMENT
List safety equipment necessities for this procedure
e.g.
Gloves (latex or nitrile)
Lab coat
Safety cabinet or similar sterile working station
Poss. fume hood
4.
RESPONSIBILITIES
Leader of Scientific programme/Head of CoE is responsible for all SOP’s for the procedures performed
in the units laboratories.
Leader of Scientific programme/Head of CoE is also responsible for making sure people using this
procedure are 1) qualified and 2) have been through the necessary training.
The user is responsible for following this SOP.
5.
EQUIPMENT, MATERIALS AND SOLUTIONS
List necessities for this procedure:
e.g.
Equipment:
Pipettes
2mL eppendorf tubes
etc.
Materials and solutions:
LB agar plates with added kanamycin (see S-SOP-003-LB)
etc.
6.
PROCEDURE: e.g Cloning of PCR products using TOPO TA/ZeroBlunt TOPO cloning
kits
Describe procedure step by step:
e.g.
We always use gloves and lab coat when working in the lab
Ligation
Reagents
Salt solution
TOPO vector
PCR product (fresh)
Total
(Full reaction)
(1 l)
(1 l)
(4 l)
(6 l)
Half reaction
0,5 l
0,5 l
2 l
3 l
1. Label the tubes of a PCR strip and add the reagents in the order stated above.
2. Carefully mix the solution by spinning it down and lightly flick the tubes
3. Incubate at room temperature for;
 20 min. for TOPO TA
 5 min. for ZeroBlunt
4. Place the tubes on ice. The tubes can be stored over night at -20ºC.
Transformation
Preparations:
 Turn on the heating block and set the temp to 42 ºC
 Place the SOC medium in room temperature
 Take out LB plater from the fridge, two for each reaction
 Thaw tubes of E.coli cells ON ICE
Version: 000
Number e.g. P-SOP-005
5.
6.
7.
8.
9.
10.
11.
Page 3 of 5
If running half reactions ~25µL of the cells must be transferred to new 2mL tube. Use modified
tips (tip cut off) when doing this.
Add 1 l (2 l) TOPO ligation mix to cells
Mix carefully by flicking the tubes, DO NOT use pipette.
Incubate on ice for 5–30 min. (Doesn’t really matter how long)
Heat shock for 30 sek. at 42C
Place directly on ice.
Add 125 l (250 l) SOC medium
Close the tubes and shake horizontally at 37C for 1h in shaking incubator.
Plating
12. Plate out 20l and 40l on two different plates. Be careful when flame sterilising the glass rods.
13. Put the plates upside down and leave them over night at 37.
14. Clone check (PCR directly on the colonies)
1.
Make a PCR master mix for the number of colonies you want to test. Use this recipe:
Reagents (cons.)
Buffer
dNTP (2mM)
T7 (5µL)
M13R (5µL)
Dynazyme II
MilliQ water
Total
Volume to 1rx
2,5µL
2,5µL
1,5µL
1,5µL
0,2µL
16,8µL
25µL
2. Aliquot the master mix in PCR tubes placed on ice or cooling-rack
3. Pick the colonies using pipette tips. Make sure you don’t get any agar, you only need to gentlt
touch the colony. Put the pipette tip in a PCR-tube.
4. When you are done picking colonies; remove the pipette tips, put the lids on and run PCR.
PCR program:
Denat.
Denat.
Anneal.
Elong.
Final elong.
Final step
7.
94C
94C
53C
72C
72C
10C
10 min.
30 sek
1 min
2 min
10 min
∞
X30
RISK ASSESSMENT1
The general risk factor of a SOP can be calculated using the part of the procedure with the assumed
highest risk factor.
The risk assessment associated with this SOP is based on the user following the precautions stated in
the step by step risk assessment below.
Describe harmful chemicals if applicable
List of chemicals and their H and P (R and S) phrases
Chemicals
Symbol
H phrases
nh
nh
none
nh= This is not classified as harmful according to the directive 67/548/EC
Risk value: S*K See risk matrix in HSE handbook, chapter 4.2
S=Likelihood: 1=Rare, 2= Unlikely, 3=Likely, 4= Higly likely and 5= Near certainty.
K=Consequence: 1= Minimal, 2=Minor, 3=Major, 4=Serious and 5=Catastrophic.
1
P phrases
nh
Version: 000
Number e.g. P-SOP-005-
Page 4 of 5
Risk assessment; step by step
Step by step risk assessment (see section 6)
Part of procedure
Unwanted scenarios
Precautions
1
Adding reagents
-
-
2
Mixing
-
-
3
Incubation at room
temp.
Place in ice
-
-
-
-
-
-
6
Adding ligation mix to
cells
Mixing
-
-
7
Incubate on ice
-
-
8
Heat shock at 42°C
-
-
9
Place on ice
-
-
10
Adding SOC medium
-
-
11
Shaking incubator
37°C
Plating
Flame sterilising
-
-
Plates contain E. coli
and antibiotics.
Burn skin, start small
fire
Wear gloves!
4
5
12
13
Store over night
-
Be careful, remove
anything that may
catch fire.
Know where nearest
fire
extinguishers/blankets
are
-
14
Clone check
-
-
Emergency
planning
Fire blankets
available in lab.
S*K
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
2*1
(green)
Emergency shower
or similar installed.
1*1
(green)
1*1
(green)
Overall risk assessment for this SOP
The overall risk is based equal the highest risk in the step by step risk assessment: e.g. here max S*K
=2*1 green hence:
If these procedures are followed there is a MINIMAL risk linked to these procedures.
Be careful when handling E-coli cells and plates with antibiotics. ALWAYS wear gloves.
8.
WASTE DISPOSAL
Describe waste disposal routines(how to dispose waste generated by this SOP)
e.g.
All plastic waste should be disposed of in the hazardous waste box (yellow).
Used and unused plates should be disposed of in the hazardous waste box or autoclaved and disposed
of as normal waste.
Number e.g. P-SOP-0059.
Version: 000
Page 5 of 5
REFERENCES
MSDSs
ECOonline.
Department of Biology HSE-handbook.
Add more referanes of needed:
Manuals for clonings kits comes with the kit or can be downloaded from Invitrogens webpage.
S-SOP-003-LB
I-SOP for fume hood in use
I-SOP for safety cabinet in use