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 4
1.
INTRODUCTION
Short description of contents and aims:
e.g.
Standard procedure for cloning of PCR products using TOPO cloning kit from Invitrogen. Specifically
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).
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.
Work should always be performed in fume hood when handling Phenol. The user must have had
necessary training in the use of fume hoods, read Procedure for the use of fume hoods.
Risk assessment conclusion based on the step-wise risk assessment in section 7 (select oneof the boxes
below)
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
3.
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.
NECESSARY SAFETY EQUIPMENT
List safety equipment necessities for this procedure
e.g.
Gloves (latex or nitrile)
Lab coat
Goggles
Postal address: UiO, Department of Biosciences, P.O. Box 1066 Blindern, N-0316 Oslo
Version: 000
Number e.g. P-SOP-0054.
Page 2 of 4
RESPONSIBILITIES
Section head/Head of CoE is responsible for all procedures performed in the units laboratories.
Section head/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.
Fill in this table when writing an S-SOP
Chemical
CAS number
Amount
NaOH
1310-73-2
xg
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. Goggles should be worn if the risk
assessment concludes that it is necessary (P-statements: P262, P280)
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.
Version: 000
Number e.g. P-SOP-0057.
Page 3 of 4
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 phrases
Chemical
NaOH
Symbol
H phrases
H314 Causes severe
skin burns and eye
damage.
nh= This is not classified as harmful according to the Regulation (EC) No. 1272/2008.
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
P280 Wear protective
gloves/protective
clothing/eye
protection/face
protection
P305+P351+P338 IF IN
EYES: Rinse cautiously
with water for several
minutes. Remove contact
lenses, if present and
easy to do. Continue
rinsing.
P310 IF exposed or
concerned: Immediately
call a POISON CENTER/
doctor
Version: 000
Number e.g. P-SOP-005-
Page 4 of 4
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
-
-
4
Incubation at room
temp.
Place in ice
-
-
5
etc
-
-
Emergency
planning
S*K
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
1*1
(green)
Overall risk assessment for this SOP
The overall risk is based on the highest risk in the step by step risk assessment above, here the
highest risk factor is S*K=1’1=1, 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.
For S-SOP’s: Is the final solution classified as hazardous according to Regulation (EC) No.
1272/2008? In that case the bottle has to be labelled accordingly.
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.
9.
REFERENCES
All MSDSs are available in ECOonline
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 safety cabinet in use