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Ligation Ligation 1. Prepare insert and linearised vector for the reaction below. Vector 50 - 100 ng Insert vector:insert ratio 1:3 10x Ligation buffer 2 µl T4 DNA Ligase 1 µl 2. Incubate at 16 ◦C overnight.   Create Date : 28 มกราคม 2551     Last Update : 28 มกราคม 2551 15:25:45 น. Counter : 281 Pageviews.   0 comment Share Tweet

Dephosphorylation Dephosphorylation 1. Precipitate DNA with 1/10 volume of NaOAC + 2 volume of EtOH or purified DNA through column. 2. Resuspend / elute DNA with 40 µl of DW then add to the reaction below. DNA 40 µl 10x CIAP reaction buffer 5 µl CIAP 0.6 µl DW 5 µl 3. Incubate at 37 ◦C for 15 minutes then at 56 ◦C for another 15 minutes 4. Add another 0.6 µl of CIAP to the reaction 5. Repeat step 3. 6. Stop the reaction with 300 µl of stop-solution 10 mM Tris HCl (pH 7.5) 1mM EDTA (pH 7.5) 200 mM NaCl 0.5% SDS 7. Incubate at 75 ◦C for 10 minutes 8. Precipitate with 2 Volume of EtOH or purify through column   Create Date : 28 มกราคม 2551     Last Update : 28 มกราคม 2551 15:57:50 น. Counter : 298 Pageviews.   0 comment Share Tweet

Restriction Enzyme Digestion Restriction Enzyme Digestion Enzyme ~ 1 – 2 U / 1 µg DNA 10x Buffer 10% of total volume 100x BSA Depending on enzyme DNA 1 – 5 µg DW Volume up to 10 – 200 µl Incubate the reaction at 37 ◦C for 1 hour to overnight   Create Date : 28 มกราคม 2551     Last Update : 28 มกราคม 2551 15:57:16 น. Counter : 268 Pageviews.   0 comment Share Tweet

Repairing 3’ or 5’ Overhanging End to Generate Blunt End Repairing 3’ or 5’ Overhanging End to Generate Blunt End 1. Digest 0.1 – 4 µg DNA with appropriate restriction enzyme 2. Purifythe required DNA band from gel after digestion if necessary 3. Add dNTP and EcoPol Buffer 4. Add 1-5 U of Klenow enzyme and incubate at RT for 30 minutes DNA x µl 10x EcoPol Buffer 5 µl 0.5 mM dNTP 1 µl Klenow 1 µl H2O 3 µl Total Volume = 50 µl 5. Stop reaction either by heating at 75◦C for 20 minutes or by adding 1 µl of 0.5 M EDTA or purify through column (preferred)   Create Date : 28 มกราคม 2551     Last Update : 28 มกราคม 2551 15:56:58 น. Counter : 295 Pageviews.   0 comment Share Tweet

Recipe ETC. LB Agar Plate Bactotryptone/Casine 10 g Yeast Extract 5 g NaCl 10 g Bacteria Agar 15 g Distilled Water to volume 1000 ml Autoclave --> Let cool to ~50 ◦C Ampicillin (50mg/ml) 1 g LB Broth Bactotryptone/Casine 10 g Yeast Extract 5 g NaCl 10 g Distilled Water to volume 1000 ml Autoclave --> Let cool to ~50 ◦C Ampicillin (50mg/ml) 1 g S.O.C Medium Bactotryptone/Casine 2 g Yeast Extract 0.5 g 1M NaCl 1 g 1M KCl 0.25 g Distilled Water to volume 100 ml Autoclave --> Let cool to RT --> Then add 20mM Mg2+ 20mM Glucose Filter medium through 0.2 µm filter, pH7.0 X-Gal Plate X-Gal (50mg/ml) 20 µl 0.1 M IPTG 50 µl H2O 30 µl Total Volume = 100 µl --> spread on LB/Amp plate OR Spread 40 µl of 50mg/ml X-Gal straight onto LB/Amp plate. 0.5 M EDTA (pH 8.0) Na2EDTA.2H2O 186.1 g DW Volume up to 1 L Bring pH to 8 with NaOH pellets (about 20 g); the EDTA will not dissolve until the pH is about right. Bring volume to 1 liter with dH2O and autoclave. TAE Buffer 50X Stock Solution Tris base 242 g Glacial acetic acid 57.1 ml 0.5 M EDTA (pH 8.0) 100 ml DW Volume up to 1   Create Date : 28 มกราคม 2551     Last Update : 28 มกราคม 2551 15:56:39 น. Counter : 204 Pageviews.   0 comment Share Tweet

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