Clontech protocol to use with column
Contents
1. a table top centrifuge Discard supernatant Resuspend pellet in 100 ul of the selection medium and spread on appropriate plates Incubate plates at 37 C for 24 hr LB amp selection only or for 36 48 hr for nutritional selection on M9 medium Typically 10 100 colonies will be seen on the plate for a successful transformation using plasmid isolated from yeast If you performed a parallel transformation using the control pUC19 DNA calculate the transformation efficiency i e cfu ug DNA The competent cells should have been transformed with an efficiency of gt 1 x 107 cfu ug Notice to Purchaser Falcon is a registared trademark of Becton Dickinson CHROMA SPIN CLONTECH and YEASTMAKER are trademarks of CLONTECH Laboratories Inc This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use CLONTECH products may not be resold modified for resale or used to manufacture commercial products without written approval of CLONTECH 2001 CLONTECH Laboratories Inc CLONTECH Laboratories Inc www clontech com Protocol PT3049 2 2 Version PR146072. YEASTMAKER Yeast Plasmid Isolation Kit Protocol at a Glance PT3049 2 Please read the User Manual before using this Protocol at a Glance This abbreviated protocol is provided for your convenience but is not intended for first time users Note that the corresponding steps may be numbered differently here than in the User Manual Resuspended yeast cells 50 ul 1 Add 10 ul lyticase 2 Incubate at 37 C for 30 60 min 3 Vortex Prespin CHROMA SPIN 1000 4 Add 10 pl SDS DEPC H 0 Column 5 min 5 Vortex optional freeze thaw and revortex 6 Load sample on prespun CHROMA SPIN 1000 DEPC H 70 Column 7 Centrifuge 5 min 8 Collect eluate Purified DNA ready for PCR and or transformation of E coli Preparation contains plasmids and yeast genomic DNA Figure 1 Flowchart for using the YEASTMAKER Yeast Plasmid Isolation Kit to prepare plasmid from yeast cells A Prepare Cell Lysates 1 Prepare yeast cultures for lysis as described in Section IV B 1 Each cell sample should be in a total volume of 50 ul 2 Add 10 ul of Lyticase solution to each tube Pipette the solution up and down repeatedly to thoroughly resuspend the colony or pellet Incubate at 37 C for 30 60 min Add 10 ul of 20 SDS to each tube and vortex vigorously for 1 min Optional Put samples through one freeze thaw cycle 20 C and vortex again oOo oa A WwW If necessary samples can be stored frozen at 20 C If sa
3. mples have been frozen vortex before using them B Purify Plasmid DNA using CHROMA SPIN 1000 DEPC H O Columns 1 Invert and vortex the CHROMA SPIN Column several times to completely resuspend the gel matrix 2 Remove the cap and break off the tip Place the column into one of the 2 ml polypropylene tubes provided 3 Centrifuge the column for 5 min at 700 x g in a swinging bucket rotor 4 Remove the column from the tube and discard the buffer PR14607 published 11 April 2001 YEASTMAKER Yeast Plasmid Isolation Kit Protocol at a Glance ONO Place the tip of the column into a clean microcentrifuge tube Carefully apply the sample to the center of the gel bed s flat surface Centrifuge the column for 5 min at 700 x g use a swinging bucket rotor The purified DNA solution is in the eluate Store at 20 C C Transforming chemically competent E coli with Plasmid Isolated from Yeast 11 12 13 O0 EO OT OO NS AE Prepare the chemically competent E coli cells or thaw them on ice Add 10 ul of yeast plasmid solution to a prechilled 14 ml Falcon tube Add 100 ul of competent cells to the tube and mix well by gently tapping the tube Incubate on ice for 30 min Transfer the tube to a 42 C water bath and incubate for 45 50 sec Chill on ice for 2 min Add 1 ml of LB or preferably SOC medium with no antibiotic Incubate at 37 C for 1 hr with vigorous shaking 250 rpm Centrifuge at 2 500 rpm for 5 min in
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