Cloning of DNA fragments

Monday, June 07, 2004

Description
This protocol describes the various steps involved in clonig a DNA fragment into a vector fro production of a plasmid via bacterial cells. This cincudes teh cDNA synthesis, PCR reaction, ligation reaction and transformation of chemically competent cells. See also protocol for mini prep and restriction endonuclease confirmation.

Procedure
CDNA synthesis:

1. Prepare in a sterile tube: Total RNA (0.1-0.5 ug), sequence specific primer (reverse, 15-20 pmol), water to volume of 11ul.
2. Incubate mix at 70 degrees for 5 mins and chill on ice.
3. Add the following in order: 5X reaction buffer 4?l, 10mM dNTP mix 2?l, RNAsin 0.5 ?l of 40U/?l, water to volume of 19 ?l.
4. Incubate at 37 degrees for 5 mins.
5. Add 40 U of reverse transcriptase.
6. Incubate at 37 degrees for 60 mins
7. Stop the reaction by heating at 70 degrees for 10 mins. Chill on ice.

PCR reaction to produce insert for cloning into vector:

1 Make up master mix as follows: 5 ?l of 10X PCR buffer, 1 ?l of 10mM dNTP mix, 2.5 ?l of 10 ?M sense and anti-sense primer, 2.5 U of pfu DNA polymerase, approximately 20 ng of DNA, make up to 50 ?l with water.
2 PCR cycle parameters as follows: Denaturation 95 degrees for 5 min, denaturation 95 degrees for 30 sec, annealing 5 to 10 degrees below Tm of primers, extension 72 degrees for 30 sec to 1 min (repeat last three steps for 25-35 cycles), final extension at 72 degrees for 7 mins, final hold at 4 degrees until ready to use the product.
3 Run 50 ul of the PCR product out on 1% agarose gel. 150-200 volts for 30 mins to 1 hour.
4 View bands using digital camera. If only one clear band present of the correct size cut it out and put it into a 1.5 ml eppindorf tube.
5 If more than one band present the PCR reaction will require some optimisation.
6 Gel purify excised band using a gel purification kit. (e.g. Sigma gel extraction kit).
7 Check concentration of eluted DNA by running a 1% agarose gel with amount markers (any DNA at concentrations of 1 ug/ul, 0.5 ug/ul, 0.1 m/ul, 0.05 ug/ul and 0.01 ug/ul). Run 1 ul of PCR product.
8 Roughly estimate the amount of DNA present in the excised bands (insert).

Vector:Insert ratio calculation:

1 Use the following equation to work out the vector:inser molar ratio: [ng vector DNA X Kb size of insert/Kb size of vector] X molar ration of insert/vector.
2 In most cases using either 1:5 or 1:10 molar ration of vector:insert should work. This requires a greater proportion of insert than vector.

Ligation reaction.

1 Set up ligation reaction as follows: Vector DNA 100 ng, Insert DNA calculated as above, T4 DNA Ligase 1 U, ligase 10 X buffer 1 ul, water to make up to 10 ul.
2 Incubate ligation reaction at 16 degrees overnight.

Transformation of competent cells.

1 Thaw aliquot of cells on ice.
2 Add 10 ng of DNA (approximately 1 ul of ligation reaction) to competent cells and mix gently by inversion, do not mix by pipetting. Incubate on ice for 30 mins.
3 Heat shock at 42 degrees for 45 seconds. Place on ice for 2 mins.
4 Add 3 ml LB or SOC media and incubate at 37 degrees for 45 mins to 1 hour with shaking at 150-250 rpm.
5 Place 100-200 ul of transformation mix onto selection plates.
6 Incubate overnight at 37 degrees.
7 The following day, pick colonies and prepare DNA stocks.

Recipes
LB media: 5g yeast extract, 10g bactotryptone, 5g NaCl, adjust pH to 7.4 with NaOH.

LB/Agar: 2.5 g yeast extract, 5 g tryptone, 2.5g NaCL, 7.5g Agar. Make up to 500 ml with water.

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Cloning of DNA fragments