Design oligos using http://helixweb.nih.gov/dnaworks/
First Step:
1. Mix oligos, 15 nM each in 20 ul total volume.
2. Add 1 mM dNTP
3. Add 1 unit of polymerase
4. Run PCR for 10 cycles

Second Step:
1. Add 2 ul first step PCR product
2. Add 1 uM F and R primer
3. Add 1 mM dNTP
4. Add 1 unit polymerase
5. Add water to 50 ul
6. Run PCR for 25 cycles

Preferred polymerases
Phusion > PfuTurbo > Pfx 50 > Pfu Ultra II

General safety precautions:
1. All laboratory personals should wear proper personal protective equipment including a laboratory coat, gloves and goggles. These items would minimize contact of samples with the skin and the eyes. Perspex goggles, especially for those who do not wear spectacles, would also serve to protect his/her’s eyes from accidental exposure to ultra-violet irradiation.

2. Never touch door handles, blue light transilluminator,  lab cameras, microscopes or telephone receivers with gloved hands.  These are surfaces that are commonly handled without gloves and come into contact with the unprotected face. As such, contamination of these surfaces pose a danger to unwary laboratory personals. Gloves must always be assumed to be contaminated with biological material. As such handling door handles and telephone receivers is always assumed to contaminate these items.

3. Clothing should extend to the ankles to protect the lower limbs. This is to ensure that any sample or liquid spill will not come into direct contact with the skin of the lower limbs.

4. No cloth or open-toed footwear should be used in this laboratory. Similarly, footwear serves as a barrier to direct contact of contaminants with the skin. However, to be effective the footwear should completely encase the feet. Sandals and slippers should never be used in this facility. Shoes made of cloth are also not effective barriers, as liquid spills could easily soak through the material. Leather or polyester shoes are most appropriate.

5. Be sure to wash your hands thoroughly before leaving the lab. Always assume that the hands have come into contact with samples, even when handling them with gloves. In order to protect laboratory personals, as well as to prevent the exit of contaminants, everybody exiting the cell culture lab must first wash his/her hands using the proper hand washing technique.

6. Never consume any food or drink inside the lab. Since the samples handled in the facility are potentially dangerous, ingestion of food and drink will increase the danger of toxins entering one’s system. As with all laboratories, food and beverage consumption is strictly forbidden in the cell culture lab. It should also be noted that the application of cosmetic products in the facility poses similar threats and should be avoided.

Safety precautions for working in the Lentiviral Production Lab:

1. Always use aseptic technique when handling biological samples. Proper aseptic technique ensures that contamination of clean surfaces is minimized if not eliminated altogether. The use of this technique not only protects your sample from contamination, it also confines and prevents your sample from contaminating the environment.

2. Using the biosafety cabinets for work that could potentially generate aerosols, such as fluid dispensing using a pipette, would minimise the contamination of the environment with these aerosols. However, for this to be effective, proper use of the biosafety cabinet must be ensured.

3. It is advisable to have yourself immunized against Hepatitis B before starting work in the lab. Since our projects often entail the use of materials derived from human sources the possibility of infection by contaminating hepatitis B viruses in these samples exists.

4. Any incidents involving a sample spill or an injury must be reported as soon as possible to facilitate treatment. This is for reasons of safety, not punishment. Contamination of the environment poses are real hazard to other laboratory personals who might not be aware of the contamination, or who might not be competent in managing such incidents. Getting help immediately would ensure that the contamination is contained and treated. Also, any injury would be attended to immediately.

5. All biohazard wastes must be double-bagged and autoclaved prior to disposal.  

Waste disposal
1. Like all laboratories, this cell culture lab will be generating considerable waste material that must be disposed of.

2. However, owing to the biohazardous nature of the materials used, special methods of disposal must also be employed for these biological wastes, in addition to the regular means of disposing non-biohazardous materials.

3. These special methods serve the following purposes:
• To minimize or remove the hazardous nature of these materials;
• To minimize the risk of laboratory personals exposure to still-hazardous wastes;
• To minimize the risk of contaminating the environment with these materials.

4. This following description of waste disposal procedures begins with general practices for common waste disposal, followed by those specific to dealing with biohazardous wastes generated from BSL-2 work.

Basic disposal protocol – General:
1. All solid non-biohazardous wastes must be discarded into the regular waste bin provided.

2. Do not discard biohazardous wastes into the regular bins or the sink.

3. Syringe needles must only be discarded in the sharps disposal bins.

4. Broken glass should not be handled manually, but should be collected using a broom and dust-pan, and then disposed of in the glass disposal box.

Basic disposal protocol – Biological:
1. All biohazardous wastes should be treated chemically or by autoclaving in order to minimize their hazardous nature prior to disposal.

2. All laboratory personals should bear in mind, however, that although chemical treatment and autoclaving are very effective methods of destroying pathogens, there is a might survive these processes and still pose a threat.

3. Treat all liquid biohazards at least 15 min with 10% (v/v) bleach before disposal into a labeled collector bottle. Do not discard into the sink.

4. All solid biohazardous wastes must be disposed of into the biohazard bins only. Do not discard into the regular waste bins.

5. All biohazard bins must be lined with biohazard bags when in use.

6. Biohazardous wastes, even when in biohazard bags, should never be brought out of the lab until properly treated, such as by autoclaving.

7. All biohazard bags should be enclosed in an additional biohazard bag before being autoclaved.

8. Discard all autoclaved waste into the collector bins provided for final disposal.

Accident management
1. Accidents can and do occur in laboratories.

2. As with all such incidences, the foremost concern is to ensure that safety to all lab personals is preserved.

3. Once all lab personals have been brought to safety, one can then begin to deal with the effects of the accident.

4. Accidents tend to generate situations of danger owing either to exposure to poisons or to physically harmful objects. These can be categorized into common dangers, and biohazardous dangers.

5. Managing these dangers requires that we first determine the nature of these dangers, and whether remaining in the site of the accident would pose a health threat.

6. If immediate evacuation is not necessary, we can then assist anyone that might have been hurt as a result of the accident. Various means of treating injuries will be dealt with later in this section.

7. Once all laboratory personals have been brought to safety, the next concern would be to prevent the threat from spreading from the site of the accident. This might necessitate the containment of spills or leaks.

8. Once the spread of the spilled biohazardous material is stopped or retarded, one may proceed to remove them and treat the contaminated surfaces.

Injuries:
1. A blunt blow to parts of the body might result in mild internal bleeding leading to a bluish-black discolouration, or a bruise, just beneath the skin.

2. In the event of a bruise apply ice cubes wrapped in a piece of cloth to the injured area. If possible, apply pressure and raise the injured part of the body to reduce swelling and bruising.

3. Cuts are dangerous because they tear the skin and make the interior of the body vulnerable to toxins and other dangerous agents.

4. If a cut is bleeding, apply pressure to it with a bandage, or with gloved hands, to staunch the flow of blood.

5. Then, the wound should be cleaned very thoroughly by rinsing with soap and water. Finally, a bandage or sticking plaster  should be used to protect the wound from exposure to the environment.

6. In the event that the cut was caused by an object known to contain or to be contaminated by a biohazardous material, immediate medical attention should be sought. If possible, seek the help of the Safety and Health Officer  or your supervisor immediately.

7. Needles are commonly used the laboratory and it is possible that a user might be accidentally stabbed with one. A needle-stick injury like this is similar to a cut and poses the same dangers.

8. Unlike a cut, a needle-stick injury may not bleed profusely and so, might not seem as severe. However, in such cases, the skin has also been penetrated and it should always be assumed that the interior of the body has been exposed to dangerous   materials. As such, needle-stick injuries should be treated the same way as cuts.

9. It should be noted however, that in the interest of safety, any object that has caused a cut, or a needle that has stabbed a person should always be assumed to have been contaminated with a biohazard. As such, the Safety and Health Officer or your supervisor should be informed and the injured person should seek medical assistance immediately.

Containment protocol:

Containing solid spills
Containing liquid spills

Basic decontamination protocol:
1. Make sure to put up clear warning signs to others to keep clear of the contaminated area until decontamination is completed.

2. In case of a solid sample spill, double-glove your hands and dispose of the contaminant, along with the outer-most pair of gloves into the biohazard bin.

3. Disinfect the contaminated surface with 10% bleach for 30 mins.

4. Wipe away the bleach, then disinfect with 70% ethanol for 30 mins.

5. Dispose of all wiping towels, along with the second pair of gloves, in the biohazard bin.

6. In case of liquid spills, prevent spread of the liquid first and foremost.

7. With double-gloved hands, use either a spill kit sponge pad or wad of paper towels to surround the liquid.

8. Proceed to soak up the main body of the spill with paper towels or sprinkle with crystalising agent (such as Red Z from the spill kit) before disposing, along with the outermost pair of gloves, into the biohazard bin.

9. Disinfect the contaminated surface with 10% bleach for 30 mins.

10. Wipe away the bleach, then disinfect with 70% ethanol for 30 mins.

11. Dispose of all sponge pads and wiping towels, along with the second pair of gloves, in the biohazard bin.

12. In case of bodily contact with a contaminant, seek help immediately from the Safety and Health Officer or your supervisor.

13. If you are alone, remove all contaminated clothing and rinse yourself thoroughly under the emergency shower. Use soap if possible.

14. In case of a minor contamination, wash the area thoroughly with soap and rinse in the sink.

15. Report all incidents of contamination to the Safety and Health Officer or your supervisor.

16. It should be noted that the reasons for reporting these incidences is solely for the purpose of ensuring the safety of the  lab personals , and not for the purpose of punishment.

Standard Operating Procedures (SOP) for Working Safely with Lentiviral Vectors

The following personal protective equipment MUST be worn when working with Lentiviral vectors:
• Gloves (consider double-gloving depending on the procedures being performed)
• Lab Coat
• Goggles
• Face shield

Special Handling Procedures
1. Cells exposed to lentiviral vectors may not be removed from the laboratory for experimental purposes unless inactivated by approved procedures.
2. If you need to aerate cultures, it must be done slowly and in a manner that minimizes the potential for aerosol creation. This action must be carried out in a class II biological safety cabinet.

3. When pouring and pipetting samples, it must be done gently and slowly and must be carried out in a class II biological safety cabinet

4. Extra precautions must be taken when using sharps. Appropriate substitutes for sharp items must be used whenever they are available. No sharps (including needles and Pasteur pipettes) may be used for working with Lentivirus-infected cell cultures nor when harvesting virus pellets. Use plastic aspiration pipettes instead of glass Pasteur pipettes.

5. For Aspiration- Use a plastic vacuum flask with a second vacuum flask connected to it as a backup, with non-collapsible tubing capable of withstanding disinfection. To the second vacuum flask attach a hydrophobic and a HEPA filter (or combination filter) to ensure that nothing is sucked into the house vacuum system. These 3 items must be attached in series from the vacuum source in the hood or a vacuum pump

Decontamination/Clean-Up Procedures
All materials that have come into contact with Lentiviral vectors should be disinfected using a 1:10 bleach solution before disposal. Additionally, all work surfaces must be disinfected with a 1:10 solution of bleach once work is completed and at the end of the work day. (Note: A 15 minute contact time is required for decontamination)

Waste Disposal Procedures
1. Non-Sharp Waste- All cultures, stocks, and cell culture materials must be disinfected and autoclaved prior to being disposed of into a double red bag-lined biohazard box.

2. Sharps Waste-All needles, syringes, razors, scalpels, Pasteur pipettes and pipette tips must be disposed of in an approved, puncture resistant sharps container. Sharps containers must not filled more that 2/3 of their capacity.

Injury/Exposure Incident Procedures
1. Eye or Mucous Membrane Exposure from Splash or Aerosols- rinse a minimum of 15 minutes using eye wash and report the incident to your supervisor immediately.

2. Skin Contamination-Wash affected areas with soap and water for 15 minutes and report the incident to your supervisor immediately.

3. Needlestick and/or Sharps Exposure- Wash affected areas with soap and water for 15 minutes. Immediately notify your supervisor. He/she will complete the Post-Exposure Incident Report and submit it to the Department of Environmental Health and Safety within 24 hours of the exposure. Contact biosafety officer to arrange for appropriate medical attention.

Spill Response Procedures
The following steps must be taken when cleaning up a spill:
1. Stop, notify others and isolate the area!
2. Put on appropriate PPE (lab coat, gloves, eye and face protection).
3. Remove glass/lumps with forceps or scoop if applicable and place into a rigid, puncture resistant container.
4. Small spills-Place paper towels soaked in bleach directly on the spill and let soak for 20 minutes.
5. Wipe up area and discard towels in biohazard waste container.
6. Continue wiping area with paper towels soaked in bleach until the spill area is completely cleaned.
7. Discard all materials in biohazard waste container.
8. Wash hands thoroughly.

ecotropic virus effectively transduces mouse or rat cells but infects human cells with extremely low efficiency. The amphotropic virus has historically been the virus of choice for infection of human and other mammalian cell lines. Recently the 10A1 envelope protein has been used due to its increased versatility relative to the amphotropic protein. The 10A1 protein recognizes the same cell-surface receptor as the amphotropic envelope protein plus a second receptor, and thus can essentially infect any cell that an amphotropic virus can infect, although in some cases with a higher efficiency. The ecotropic, amphotropic and 10A1 proteins are all natural MMLV variants, and are all relatively labile and thus considered relatively safe compared with other viral systems. The vesicular stomatitis virus G protein (VSV-G) is rapidly becoming the most popular envelope protein. Unlike the other three MMLV-derived envelope proteins which recognize cell surface receptors, VSV-G recognizes a phospholipid that is present on all cell types, and thus can theoretically allow the efficient infection of any mitotic cell.

Buffer Preparations:
Buffer A:
Triethylamine acetate (TEAA) 1M (pH 7.0) 200 ml;
Tris/HCl 1 M (pH 7.5) 200 ml;
adjust volume to 1000 ml with dH2O, pH 7.5;

Buffer B:
10 x STE (0.2M Tris-HCl, 0.01M EDTA, 0.05M NaCl, pH 8.0) 100 ml;
Ethanol 20 ml;
adjust volume to 1000 ml withh dH2O

Buffer C:
Sodium acetate anhydrous salt (FW = 82.03 g) 8.2 g;
Ethanol 250 ml;
adjust volume to 1000 ml with dH2O; pH 8.5.

1. Pack Perfluorosorb S column
2. Equilibrate column with Buffer C.
3. Dissolve plasmid DNA in Buffer C.
4. Load  sample onto the equilibrated column.
5. Wash the column with 23 column volumes of Buffer C to remove unbound impurities.
6. Wash column with 5 column volumes of Buffer B to remove loosely bound impurities.
7. Elute bound DNA with Buffer A (4 column volumes).