Mutagenesis with In-Fusion® HD Cloning Plus
Contents
1. 638909 In Fusion HD Cloning Plus 10 Rxns 638910 Liquid system includes the In Fusion Enzyme CloneAmp HiFi 50 Rxns 638920 PCR Premix Stellar Competent Cells and NucleoSpin Geland 96 Rxns 638911 PCR Cleanup Kit 100 Rxns Notice to Purchaser Your use of these products and technologies is subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories Inc Clontech2. Part VI Protocol I A in the e PrimerT s should be 58 65 C Difference be In Fusion HD Cloning Kit User Manual he ale and reverse primerT_s should 6 Assemble the In Fusion reaction e lt 4 C 7 i Linear construct containing 100 ng 3 Prepare CloneAmp HiFi PCR Master Mix your mutation CloneAmp HiFi PCR Premix 12 5 ul In Fusion enzyme 2 ul Forward primer 200 300 nM H O As needed Reverse primer 200 300 nM Template 0 1 5 0 ng Totalvolime 10 pl H O As needed 7 Incubate the reaction at 50 C for 15 min Totali vol rne rxi 25 ul 8 Transform Stellar Competent Cells with 2 5 ul of the In Fusion reaction 4 Linearize the vector by inverse PCR using a three 9 The next day screen for mutants You have a gt 95 step PCR protocol and CloneAmp HiFi PCR Premix chance of recovering your final desired construct 30 35 cycles the very first time 98 C 10 sec 55 C 5 or 15 sec Reference 72 C 5 sec kb 1 Ochman H Gerber A S Hartl D L 1988 Genetics 120 3 621 623 Cat Product Package Size 638916 In Fusion HD Cloning Plus CE 10 Rxns 638917 Liquid system includes the In Fusion Enzyme CloneAmp HiFi 50 Rxns 638919 PCR Premix Stellar Competent Cells and Cloning Enhancer 96 Rxns 638918 100 Rxns 638912 In Fusion HD EcoDry Cloning Plus 8 Rxns 638913 Lyophilized system includes the In Fusion Enzyme CloneAmp 24 Rxns 638914 HiFi PCR Premix Stellar Competent Cells and Cloning En 48 Rxns 638915 hancer 96 Rxns
3. CLONING AND COMPETENT CELLS Mutagenesis with In Fusion HD Cloning Plus e Asingle system for deletions base substitutions or additions Timeline l e Flexible enough to use with any vector Before you begin e Over 95 efficiency guaranteed Pick your vector any vector PRS and envision In Fusion PCR cloning makes it easy to perform mutagenesis it combines the ie power of the In Fusion HD enzyme with inverse PCR a method for rapid in vitro amplification of the DNA sequences that flank a region of known sequence 1 During inverse PCR primers are oriented in opposite directions on your circular cloning vector Figure 1 To perform mutagenesis with In Fusion systems design your PCR primers so that they have a 15 bp overlap with each other Day One at their 5 ends and incorporate the mutation of interest Use the CloneAmp HiFi PCR Premix a high fidelity PCR polymerase included with all In Fusion HD O Cloning Plus Systems to perform your PCR reaction add the In Fusion HD PCR using enzyme premix to your linearized PCR product and transform into the provided Design and order primers Q Linearize eae Stellar competent cells You have a gt 95 chance of recovering your final E desired construct the first time and every time In Fusion F i reaction Experimental Overview a 1 Think about your final construct Choose the vector you want to modify and product envision your final mut
4. after the In Fusion reaction Similarly if you would like to change one or more bases in a construct design primers that include 15 bp overlaps with each other and contain the desired substitutions within the overlapping region Figure 5 Added or substituted bases go here Reverse primer XXX xx Forward A primer Changed bases are within 15 bp overlap Le Ld Le E 1 Envision final 2 Design 3 Amplify linear 4 Recover final construct primers construct construct Figure 5 In Fusion primer design for inserting or substituting bases Primers are designed to add additional bases or to replace existing bases with different bases Xs in the in the primers and the final vector in the original vector Clontech CLONING AND COMPETENT CELLS In Fusion Mutagenesis Protocol Overview Please see the In Fusion HD Cloning Kit User Manual for detailed instructions 1 Select your vector and identify the mutation site 5 Treat the PCR product with Cloning Enhancer to remove the circular double stranded template from the reaction Refer to Part VII Protocol II A in the In Fusion HD Cloning Kit User Manual 2 Design PCR primers as described above keeping in mind these general guidelines e Primers should be 18 25 bases long Insertions may require longer primers e Primers should be 40 60 GC If your PCR product contains multiple bands gel purify instead using the NucleoSpin Gel and PCR Clean Up Kit Refer to
5. ated construct Figure 1 mutation shown in yellow Transform 2 Design your primers Design inverse primers that overlap each other by 15 bp BRE at their 5 ends and incorporate your desired deletion substitution or addition Stellar com Specific guidelines for mutagenesis primer design are described below aa 3 Utilize the power of In Fusion Using an inverse PCR protocol amplify Day Two the vector with your new primers Perform the In Fusion reaction using Recover the PCR product The linear DNA will re circularize at the site of the 15 bp your final overlap and will also contain your mutagenic changes Transform a portion construct of the In Fusion reaction into Stellar Competent Cells according to the In Fusion HD Cloning Plus protocol 4 Obtain your final construct Recover your mutant from the Stellar cells Note Although all the examples the following day shown here involve protein coding gene sequences you can use Change occurs the same methods to modify non here coding sequences such as promoters I or transcription factors O l lly Visit our website EE EE 15 bp O Ayy overlap C O EEE L J L L L J 1 Envision final 2 Design 3 Perform In Fusion protocol 4 Recover final construct primers e Amplify linear construct construct e Perform In Fusion reaction to re circulize vector at 15 bp overlap e Transform into Stellar Competent Cells Figure 1 Procedure for performing mutagene
6. of turquoise Note that there is no gap between the pink and turquoise regions in the actual primer sequence the deleted nucleotides are not included in either of the primers Deletion site J Reverse primer Forward primer 15 bp overlap Ll L_a Ld 1 Envision final 2 Design primers then 3 Recover final construct perform In Fusion construct protocol Figure 2 In Fusion primer design for deletion mutagenesis Primers are designed to eliminate a section of the original vector To create a series of C terminal deletions design only one forward primer that anneals to your cloning vector immediately downstream of the coding region retaining the stop codon Then design a series of reverse primers that include 15 bp of overlap with the forward primer at their 5 ends and span different regions to be deleted In Figure 3 Construct A has the blue region deleted Construct B has the blue and turquoise regions deleted and Construct C has the blue turquoise and pink regions deleted Deletion Deletion Deletion site sites sites J Forward N Reverse primer primers N Ata B42 Original cH vector A 15 bp overlap Ll 1 Envision final constructs 2 Design primers 3 Recover final constructs then perform In Fusion protocol Figure 3 In Fusion primer design to create a series of C terminal truncated proteins Primers are designed to eliminate one or more contiguous sections of the original vector Clon
7. sis with In Fusion systems The area where mutagenesis occurs is shown in yellow r United States Canada 1 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 724 Clontech Laboratories Inc For Research Use Only Not for use in diagnostic or therapeutic procedures Not for resale Clontech the Clontech logo CloneAmp ATakara Bio Company EcoDry In Fusion and Stellar are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions 2013 Clontech Laboratories Inc Clontech 6 13 IN 633337 www clontech com CLONING AND COMPETENT CELLS Primer Design for Deletion Mutagenesis Primer design is a key component of simple In Fusion based deletion mutagenesis To delete a region of your cloning vector you must design primers that include 15 bp overlap with each other at their 5 ends and do not include the bases to be deleted Figure 2 For visual interest and easy understanding of the primer design concept different regions of the vector backbone and primers are marked in color In Figure 2 the deletion site is marked in yellow and the binding site for the reverse primer pink and turquoise spans the deletion The binding site for the forward primer turquoise and black is located against the cloning vector backbone The two primers overlap by 15 bp at their 5 ends the common area
8. tech CLONING AND COMPETENT CELLS To create a series of N terminal deletions design one reverse primer that anneals to your cloning vector immediately upstream of the coding region retaining the stop codon Then design a series of forward primers that retain the natural start codon include 15 bp of overlap with the reverse primer at their 5 ends anneal to the coding region you wish to maintain at their 3 ends and span different deletions In Figure 4 Construct D has the turquoise region deleted and Construct E has the turquoise and blue regions deleted Deletion Deletion i Al Forward primers Reverse ND a NE Original vector 15 t overlap Lad 1 Envision final 2 Design primers 3 Recover final constructs then perform constructs In Fusion protocol Figure 4 In Fusion primer design to create a series of N terminal truncated proteins Primers are designed to eliminate one or more contiguous sections of the original vector Primer Design for Base Insertions or Base Substitutions Inserting bases is simple with the In Fusion systems To insert bases design primers that include 15 bp overlaps with each other at their 5 ends and contain the desired insertion s within the overlapping region Figure 5 Only the 15 bases at the 5 ends of the primers are required to overlap but depending on the length and sequence of your insertion the overlap may be longer than 15 bp Additional bases added to the primer will be maintained
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