Protocols

Miniprep Prodedure

• The purpose of this procedure is to extract plasmid DNA and separate it from chromosal DNA in a cell. All DNA (chromosal and plasmid) is denatured in the first step. Plasmid DNA, unlike chromosomal DNA, forms linked rings upon denaturation. Next, DNA is renatured with the use of buffers. Chromosomal DNA remains denatured because of the inefficiency of re-ligation of multiple pieces and strands of DNA. The plasmid DNA renatures and is thereby, separated from chromosomal DNA.
• Before starting the miniprep, the following steps should be completed with a new miniprep kit.

1. Add RNase to Buffer P1 and store in the refrigerator.
2. Add ethanol to Buffer PE.
3. Check Buffers P2 and P3 for salt precipitation and redissolve at 37 degrees C is neccessary.

• After preliminary steps have been done the miniprep is performed with the following steps:

1. 2:30 PM: Resuspend the pelleted bacterial cells in 250 microliters Buffer P1 and put in a centrifuge tube.
2. Add 250 microliters Buffer P2 and mix, invert 4-6 times. Solutions containing LyseBlue will turn blue.
3. Add 350 microliters N3 and IMMEDIATELY invert 4-6 times. Solutions with LyseBlue will turn colorless.
4. Centrifuge for 10 minutes at 13,000 rpm.
5. Apply supernatant to QIAprep spin by decanting or pipetting.
6. Centrifuge for 30-60 seconds at 13,000 rpm. Discard flow through.
7. Wash QIAprep spin column. Add .5 mL PB. Centrifuge 30-60 seconds.
8. Wash QIAprep spin column by adding .75 mL PE. Centrifuge 30-60 seconds.
9. Dsicard flow through. Centrifuge 1 minute.
10. To elute DNA, place QIAprep column in clean 1.5 mL centrifuge tube. Add 50 microliters EB or water to center of each QIAprep spin column, making sure to pipette EB right onto the cotton plug. Let stand for 1 minutes. Centrifuge for 1 minute.

Restriction Digest

• The following procedure makes a 50ul reaction.
• Add the following:

1. Milli-Q Water (42.5 ul - Amount of DNA}
2. 5 ul Buffer
3. .5 ul BSA
4. X ul DNA (Should aim to have 500 ng if just running a gel for verification purposes. More might need to be used for gel extractions)
5. 1 ul of each restriction enzyme

• Place in the Thermocycler for 100 at 37 deg C. Heat shock at 80 deg C for 20 minutes.

Gel Electrophoresis

• Gel Electrophoresis is primarily used to determine the length of a fragment of DNA. DNA Ladders are run beside experimental fragments as points of reference. A DNA Ladder contains all different lengths of DNA that when run out on a gel, separate to reveal the number of base pairs in each gene.

1. Prepare the casting gel: 1% agarose gel - 1) 0.5 g agarose 2) 50 mL TBE + SYBR safe.
2. Gel must be submerged completely with 300-500 mL (large gel tray) .5X TBE + SYBR safe buffer.
3. Put the gel, WITHOUT the lid, in the microwave on HIGH for 1 minute.
4. Cool the bottle of gel under water.
5. Pour into the castings with rubber stoppers.
6. The gel takes approximately 20 minutes to set. Once gel is set make sure to remove the rubber stopprs.
7. DNA has a negative charge. The wells of the gel are aligned with on the negative side (Cathode). DNA will move away from the Cathode to the Anode (+).

• The gel acts as a sieve for fragements as small as 100 base pairs and as large as 50,000 base pairs. 0.3% large fragments - 2% large fragments.

1. When complete, pour TBE buffer back into a container. The buffer can be used up to 8 times.

Ligation Reaction Procedure

1. Thaw T4 DNA ligase and 10x Buffer on ice.
2. In a sterile tube add (in this specific order): 1. 8 microliters dd water 2. 1 microliter vector 3. 3 microliters insert 4. 1.5 microliter 10x Ligase Buffer 5. 1.5 microliter T4 DNA ligase
3. 2:45 PM 2 Hour Benchtop Ligation (products are kept at room temperature between 2-24 hours). When performing a short incubation, the concentration of ligase should be increased. Normally 1 microliter of T4 DNA ligase is used. Here we are using 1.5 microliters. If not proceeding onto transformation after the incubation period is complete, the ligation reaction can be stopped by freezing at -20 deg C.

• It is important to note here the 3:1 ratio used when combining the vector and insert for the ligation reaction. The ratio is in terms of molarity NOT volume. Depending on the size of the insert, the volume of insert used will vary. With a very small insert, the ratio of insert to vector might increase to around 10:1.
• The following equation can be used: Insert mass in nanograms = 3 x [Insert length (bp) / Vector length (bp)] x Vector Mass in nanograms

Transformation Procedure

1. Place both in ice. The control in this experiment is a negative control because no DNA is added to the competent cells.
2. Obtain 100 microliters of competent cells for each centrifuge tube. Competent cells must thaw before being used.
3. 3:35 PM: 4 microliters of ligation product added to competent cells and left in ice for 20 minutes.
4. 3:55 PM: Both control and DNA centrifuge tubes are placed in a 42 degree C hot bath or thermocycler for 60 seconds.
5. Both tubes are placed back in the ice for two minutes.
6. Add 900 microliters of ice cold antibiotic free LB to both centrifuge tubes.
7. 4:10 PM: Cells are incubated in 37 degree C for 60-90 minutes.
8. 5:20 PM: Three solutions are plated on LB ampicillin resistant plates. Spreader dipped in ethanol and passed through a flame to ensure sterilization before plating. The three plates contained: Plate 1) 100 microliters of the control Plate 2) 100 microliters of LB/cells (1:1 ratio) Plate 3) Dilution of 99 microliters water and 1 microliter of LB/cells (100:1 ratio)
9. Plates are incubated upside down for 24 hours in 37 degrees C.

Ethanol Precipitation

• Ethanol Precipitation is used to purify small fragments of DNA and increase DNA concentration. Since only 10 microliters of DNA can be placed in Gel Electrophoresis wells, the amount must be extremely concentrated.

1. 4:15 PM: Add 1) 3 volumes of COLD 100% EtOH (50 microliters X 3 = 150 microliters) 2) 1/10 3M Sodium Acetate (20 microliters) 3) 1 microliter GlycoBlue
2. 4:35 PM: Incubate for 1 hour at -80 deg. C
3. 5:40 PM: Centrifuge 30 minutes at 0 deg. C. A blue pellet should be visible once finished centrifuging. Pellets will dissolve if in room temperature too long before performing next step!
4. Remove supernatant.
5. Air dry.
6. Resuspend in water.

Gel Extraction

1. Excise the DNA fragment from the gel after performing gel electrophoresis.
2. Weigh the cut pieces of gel and add 3 Volumes of Buffer QG to 1 Volume of gel (1 mg is approximately 1 microliter).

• .2955 grams (mass of gel cut from gel) x 3 = (.8865 grams) x 1000 mg = 886.5 mg = 886.5 microliters (volume of Buffer QG added)

1. 2:50 PM Incubate in 50 deg C for 10 minutes to dissolve the gel completely. Every 2-3 minutes during incubation, mix contents by vortexing.

The contents of the tube should be yellow after incubation.

1. 3:00 PM Add 1 gel Volume of isopropanol. The addition of isopropanol will increase the yield of the fragements. It is used for fragements smaller than 500 base pairs of greater than 4 kb. It is optional for fragments in between 500 bp and 4 kb.
2. Place a QIAquick spin column in a provided 2 mL collection tube.
3. To bind the DNA, apply sample to QIAquick column and centrifuge for 60 seconds. The maximum volume for the spin column is 800 microliters.
4. Discard flow through. Place QIAquick column into the same collection tube.
5. Recommended step: Add .5 mL Buffer QG and centrifuge for 60 seconds.
6. To wash the DNA, add .75 mL Buffer PE and centrifuge for 60 seconds.
7. Discard flow through. Centrifuge for 60 seconds at 13,000 rpm.
8. Place column into a clean 1.5 centrifuge tube.
9. Add 34 microliters Buffer EB or water to center of the QIAquick membrane. Centrifuge for 60 seconds.
10. Add loading gel if a gel analysis will be performed after gel extraction.

Calf CIP - Calf Intestinal Alkaline Phosphatase

• This procedure is only done to the vector plasmid. Calf CIP hydrolyzes the 5 prime end of the vector (phosphate group) so it cannot religate with itself.

1. Elute gel extract in 30 microliters 10 mM Tris-HCl Buffer (Buffer EB)
2. CIAP 10x Reaction Buffer and CIAP enzyme should thaw on ice until used. Add 4 microliters CIAP 10x Reaction Buffer and 1 microliter CIAP enzyme to the 34 microliters from the gel extraction. The total volume should be 39 microliters.
3. 4:10 PM Incubate in 37 deg C for 30 minutes.
4. 4:30 PM Add 1 microliter CIAP enzyme. Incubate in 37 deg C for 30 minutes.

• An Ethanol Precipitation is performed on the vector after the Calf CIP procedure and on the insert after Gel Extraction. The Ethanol Precipitate is usually placed in -80 deg C overnight OR for 1 hour coupled with a 30 minute centrifuge at 4 deg C.