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List of Commonly Used Lab Buffers

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol lists out the commonly used lab buffers and which protocol they are used in.

Protein Purification Buffers

Freestyle Media

The FreeStyle Media is used to culture the HEK293f cells used in Protein Production. This media is ordered and is normally stored in 1L plastic bottles in the Cold Room. Bottles that are actively being used are stored in Eman's shelf of the Main Lab Fridge next to the Weigh Scale.

Bottles that are actively being used should be wrapped with Aluminum Foil to prevent UV-Degradation.

No additives are required before using this Buffer to Culture HEK293f cells, but the buffer should be warmed in the hot water bath before being used.

Protocol(s) of Use

5M Imidazole

A reagent that is needed for the creation of other Protein Purification Buffers. This buffer is stored in the cold room and wrapped in aluminum foil.

  • 170.2g of imidazole
  • Dissolve in DI-H20 so that the Total Volume is 1000mL
  • Sterilize by submitting the solution for Autoclaving.

Do Not just immediately dissolve the imidazole in 1000mL of DI-H20 because that ignores the volume that the imidazole occupies. Dissolve in a smaller volume of DI-H20, and increase the amount of DI-H20 until the total volume reaches 1000mL

5M NaCl

A reagent that is used to stabilize the protein supernatant during Protein Purification. This buffer is stored at room Temperature by Eman's Bench or in the Cold Room.

  • 292g of NaCl
  • Dissolve in DI-H20 so that the Total Volume is 1000mL
  • Sterilize by filtering with a 0.22um Vacuum Filter

Do Not just immediately dissolve the NaCl in 1000mL of DI-H20 because that ignores the volume that the NaCl occupies. Dissolve in a smaller volume of DI-H20, and increase the amount of DI-H20 until the total volume reaches 1000mL

Protocol(s) of Use

50mM EDTA

A reagent used in the process of regenerating Ni-Resin. This buffer can be stored either at room temperature at Eman's Bench or in the cold room.

  • 100mL of 0.5M EDTA
  • 900mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

Protocol(s) of Use

0.5M NaOH

A reagent used in the process of regenerating Ni-Resin. This buffer can be stored either at room temperature at Eman's Bench or in the cold room.

  • 20g of NaOH
  • Dissolved in 1000mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

Be careful as the reaction for dissolving NaOH in H20 is exothermic so the glass will heat up as you dissolve the NaOH.

Protocol(s) of Use

100mM NiSO4

A reagent used in the process of regenerating Ni-Resin. This buffer should be stored in the cold room.

  • 26.3g of NiSO4 • 6H20 (molecular weight 263 g /mol)
  • Dissolve in a total of 1000mL of DI-H20
  • Sterilize by filtering with a 0.22um Vacuum Filter

Do Not just immediately dissolve the NiSO4 • 6H20 in 1000mL of DI-H20 because that ignores the volume that the Nickle occupies. Dissolve in a smaller volume of DI-H20, and increase the amount of DI-H20 until the total volume reaches 1000mL

Protocol(s) of Use

20% ETOH

A reagent used to mix with the Nickle Resin to create a slurry suitable for storage. This buffer should be stored in the cold room. Any slurry created from this buffer should also be stored in the cold room.

  • 200mL of 100% ETOH
  • 800mL of DI-H20

Protocol(s) of Use

10X Wash Buffer

A reagent used in Nickle Column Chromatography. Specifically, this is used to make the 1X variant. This buffer should be stored in the cold room and wrapped in aluminum foil.

  • 250mL 5M NaCl
  • 10mL of 5M Imidazole
  • 240mL of PBS

Protocol(s) of Use

10X Elution Buffer

A reagent used in Nickle Column Chromatography. Specifically, this is used to make the 1X variant. This buffer should be stored in the cold room and wrapped in aluminum foil.

  • 250mL 5M NaCl
  • 250mL 5M Imidazole

Protocol(s) of Use

1X Wash Buffer

The reagent actually used in Nickle Column Chromatograph. This should be stored in the cold room and wrapped in aluminum foil.

  • 55mL of 10X Wash Buffer
  • 500mL of PBS

Protocol(s) of Use

1X Elution Buffer

The reagent actually used to Elute the Protein during Nickle Column Chromatography. This should be stored in the cold room and wrapped in aluminum foil.

  • 55mL of 10X Elution Buffer
  • 500mL of PBS

Protocol(s) of Use

NHP PB / BM Buffers

Hemolytic Buffer

This reagent is used to lyse the RBCs from a sample of PB or BM to isolate only the WBCs. This should be stored in either the cold room, or in the TC Room Fridge if being used.

  • 32.1 g of Ammonium Chloride
  • 4.03 g of Sodium Bicarbonate
  • 0.8 mL of 500mM EDTA
  • 4L of Water
  • Sterilized by filtering with a 0.22um Vacuum Filter

The recipe can be scaled down depending on the volume of Hemolytic Buffer you need. Most commonly, this recipe is made in 1L (Divide everything by 4) or in 500mL (Divide everything by 8).

Protocol(s) of Use

MACS Buffer

This reagent is used to keep cells undergoing a CD34+ Enrichment stable and happy. This should be stored in either the cold room, or in the TC Room Fridge if being used.

  • 2.5g of Bovine Serum Albumin
  • 2mL of 0.5M EDTA
  • 500mL PBS
  • Sterilized by filtering with a 0.22um Vacuum Filter

Protocol(s) of Use

Cell Culture Buffers

DMEM++ Buffer

This buffer mix is used for culturing HEK293 T Cells. Specifically for use in virus prep. This should be stored in the TC Room Fridge.

  • 500mL DMEM Media
  • 50mL Heat Inactivated Cosmic Calf Serum
  • 5.5mL of PenStrep
  • 5.5mL of Sodium Pyruvate (100X)
  • 5.5mL of Non-Essential Amino Acids (100X)
  • 5.5mL of L-Glutamine (100X)
  • Sterilized by filtering through a 0.22um Vacuum Filter

Protocol(s) of Use

IMDM++ Buffer

This buffer mix is used for culturing LLCMK2 cells. This should be stored in the TC Room Fridge.

  • 500mL IMDM Media
  • 50mL of Heat Inactivated FBS
  • 5.5mL of PenStrep
  • 5.5mL of L-Glutamine (100X)
  • Sterilized by filtering through a 0.22um Vacuum Filter

Protocol(s) of Use

RPMI w/ HEPES

This buffer mix is used for culturing Kasumi Cells. This should not be mistaken for the media used for culturing Jurkat Cells. This should be stored in the TC Room Fridge.

  • 500mL RPMI 1640 w/ HEPES
  • 50mL of Heat Inactivated FBS
  • 5.5mL of PenStrep
  • 5.5mL of L-Glutamine (100X)
  • Sterilized by filtering through a 0.22um Vacuum Filter

Protocol(s) of Use

RPMI w/o HEPES

This buffer mix is used for culturing Jurkat Cells. This should be stored in the TC Room Fridge.

  • 500mL of RPMI 1640 w/o HEPES
  • 50mL of Heat Inactivated FBS
  • 5.5mL of PenStrep
  • 5.5mL of L-Glutamine (100X)
  • Sterilized by filtering through a 0.22um Vacuum Filter

Protocol(s) of Use

Freeze Media

This buffer is used to freeze down cells for liquid nitrogen storage. The recipe below is for the 1X solution. Scale the recipe up depending on how many samples you have. Always make more than strictly necessary to account for pipetting errors.

  • 900mL of Heat Inactivated FBS
  • 100mL of DMSO

Protocol(s) of Use

Thaw Media

This buffer is used to recover cells from liquid nitrogen storage. The recipe below is buffer to thaw 1 sample. Make each aliquot of this media in it's own 50mL Centrifuge Tube. This buffer should be kept on ice while being used.

  • 31.5mL of Heat Inactivated FBS
  • 3.5mL of DNase I

Protocol(s) of Use

Stem Cell Media

This buffer is used to culture stem cells collected after a CD34+ Enrichment. The buffer should stored in the TC Room Fridge and is stable for up to 1 month.

  • 50mL SFEMII Media
  • 500uL of PenStrep
  • 50uL of TPO Cytokine
  • 50uL of SCF Cytokine
  • 50uL of FLCT3 Cytokine
  • Sterilized by filtering through a 0.22um SteriFlip Filter

Protocol(s) of Use

Misc Buffers

0.1% Gelatin

This reagent is used to coat the plates during a viral transfection. It should be stored in the TC Room Fridge but should be warmed in the hot bath prior to use to ensure all the gelatin has dissolved properly.

  • 50mg of Gelatin
  • Dissolved in 50mL of DI-H20
  • Solution should be left in the hot water bath and vortexed periodically to allow for the gelatin to completely dissolve

Protocol(s) of Use

LV-Max Production Media

This reagent is used 24 hours after adding the transfecting plasmids during a virus prep. It should be stored in the 4C fridge or the cold room.

  • 1000mL of LV-Max Production Media
  • 20mL of 1M HEPES
  • 10mL of PenStrep
  • 10mL of L-Glutamine (100X)

Protocol(s) of Use

2% Gel Agarose Solution

This reagent is used to cast DNA Electrophoresis Gels. It should be stored at room temperature on the shelves above the Gel Bench. The solution should be microwaved to allow the solution to melt before being used.

  • 3.6mg of UltraPure Agarose Powder
  • 200mL of 1X TAE Buffer
  • 100mL of GelRed Nucleic Acid Stain
  • Solution should be mixed by swirling and then microwaved for 3 minutes. The Solution should be periodically swirled every minute to help the agarose dissolve. The solution should appear translucent when the agarose has completely dissolved.

Protocol(s) of Use

2% BSA Solution

This reagent is used for retronectin coating plates. The solution should be kept in the TC Room Fridge.

  • 1g of Bovine Serum Albumin
  • 50mL of PBS
  • Sterilized by filtering through a 0.22um SteriFlip Filter

Protocol(s) of Use