Produce and Collecting Ascitic Fluid from Mice

Ascitic fluid (also called ascites) is an intraperitoneal fluid extracted from mice that have developed a peritoneal tumor. For antibody production, the tumor is induced by injecting hybridoma cells into the peritoneum, which serves as a growth chamber for the cells. The hybridoma cells grow to high densities and continue to secrete the antibody of interest, thus creating a high-titered solution of antibodies for collection. Antibody concentrations will typically be between 1 and 10mg/ml.

1. Prime adult female mice (at least 6 weeks old) of the same genetic background as your hybridomas by injecting 0.5mL of pristane (2,6,10,14-tetramethyldecanoic acid) into the peritoneum. These solutions will act as irritants to the mice, which respond by secreting nutrients and recruiting monocytes and lymphoid cells into the area. This creates a good environment for the growth of the hybridoma cells.
2. After 7-14 days, inject 5x10(5) to 5x10(6) hybridoma cells ip. Prior to injection, the cells should be growing rapidly. Centrifuge the cells and wash once in PBS. Inject the cells in no more than 0.5mL of PBS.
3. Ascitic fluid may begin to build up within 1-2 weeks following the injection of the cells. Tap the fluid when the mouse is noticeably large, but before the mouse has difficulty moving. Carefully withdraw as much fluid as possible with 18-gauge needle attached to a 5mL syringe.
4. Return the mouse to its cage. Many mice will produce a second or third batch of ascitic fluid. You can also bleed out the mouse and combine the blood with the ascitic fluid.
5. Incubate the fluid at 37°C  for 1 hour. Transfer to 4°C overnight.
6. Spin the fluid at 3000g for 10min. If there is an oil layer, remove this first and discard. Carefully remove the supernatant from the cell pellet. Spin again if necessary.

A single mouse may yield as much as 10mL of ascitic fluid per batch. Antibody concentrations may be as high as 10mg/mL.

Protocol for Real-Time PCR

This protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green as was mentioned in Xiaowei Wang and Brian Seed (2003) A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research 31(24): e154; pp.1-8. Please refer to this paper and the PrimerBank Help page for more background information. The procedure begins with reverse transcription of total RNA. The cDNA is then used as template for real-time PCR with gene specific primers. You may need to modify this protocol if you use different reagents or instruments for real-time PCR.

Time required

	cDNA synthesis: 2 hours.
	real-time PCR: 2 hours.
	Dissociation curve analysis: 0.5 hour.

DNA Precipitation

Overview

This protocol can be used to concentrate DNA, or to change the buffer the DNA is suspended in. It can also be coupled with phenol chloroform extraction for the purifying nucleic acids. This protocol also works for RNA precipitation (take care to use RNAse free materials in this case).

Materials

• 3M NaOAc pH 5.2
• EtOH 95%
• Glycogen (optional)

DNA Precipitation protocol (2)

Solutions/reagents:

• 3M Sodium Acetate solution
• Glycogen
• 95% EtOH
• 70% EtOH
• water
• buffer
• DNA sample

Equipment:

• Centrifuge

Steps:

1. Measure out 0.1 volume 3M Sodium Acetate solution into DNA sample.
2. Measure out 1 µl of Glycogen into Eppendorf tube (1).
3. Add 2 volumes 95% EtOH.
4. Option 1: Store at -20°C for 12 hrs(overnight).
   (or)
   Option 2: Store at -80°C for 30 mins.
5. Centrifuge at maximum speed for at least 15 mins at room temperature, gently aspirate out the supernatant and discard it.
(Optional)
Add 1 ml of 70% EtOH.
Store at room temperature for 5 mins.
(Optional)
Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.

6. Dry the pellet in air for 10 - 15 mins.
   Dry until all the liquid is gone.
7. Option 1: Add water to pellet.
   (or)
   Option 2: Add buffer to water.
   Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 25 mins

LAMP - Loop Mediated Isothermal Amplification

LAMP - Loop Mediated Isothermal Amplification
"LAMP" stands for Loop-mediated Isothermal Amplification. This technology was developed by Notomi et al. It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using a strand displacement reaction.

Types of Primers used in LAMP

LAMP is characterized by the use of 4 different primers specifically designed to recognize 6 distinct regions of the target gene. The four primers used are as follows:
1. Forward Inner Primer (FIP): The FIP consists of a F2 region at the 3'end and a F1c region at the 5'end. The F2 region is complementary to the F2c region of the template sequence. The F1c region is identical to the F1c region of the template sequence.
2. Forward Outer Primer (FOP): The FOP (also called F3 Primer) consists of a F3 region which is complementary to the F3c region of the template sequence. This primer is shorter in length and lower in concentration than FIP.
3. Backward Inner Primer (BIP): The BIP consists of a B2 region at the 3'end and a B1c region at the 5'end. The B2 region is complementary to the B2c region of the template sequence. The B1c region is identical to the B1c region of the template sequence.
4. Backward Outer Primer (BOP): The BOP (also called B3 Primer) consists of a B3 region which is complementary to the B3c region of the template sequence.

Multiplex PCR

Introduction of Multiplex PCR

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

Types of Multiplex PCR

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction
This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.
2. Multiple Template PCR Reaction
It uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

NASBA Technology

Introduction to NASBA

RNA detection is commonly done using RT-PCR, a time consuming process often resulting in false positives due to cross contamination. Alternatively, nucleic acid sequence-based amplification (NASBA) is a one step isothermal process for amplifying RNA. NASBA has proven to be successful in detection of both viral and bacterial RNA in clinical samples.
A NASBA reaction consists of avian myeloblastosis virus (AMV), reverse transcriptase (RT), T7 RNA polymerase and RNase H with two oligonucleotide primers. The amplification is more than 1012 fold in 90 to 120 minutes. The amplification of ssRNA is possible only when the denaturation of dsDNA does not occur. As NASBA is an isothermal process, it is thus possible. The NASBA reaction does not get false positives caused by genomic dsDNA, as in the case of RT-PCR.

PCR Primer Design Guidelines

PCR (Polymerase Chain Reaction)

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks.

PCR involves the following three steps: Denaturation, Annealing and Extension. First, the genetic material is denatured, converting the double stranded DNA molecules to single strands. The primers are then annealed to the complementary regions of the single stranded molecules. In the third step, they are extended by the action of the DNA polymerase. All these steps are temperature sensitive and the common choice of temperatures is 94^oC, 60^oC and 70^oC respectively. Good primer design is essential for successful reactions.