Engineering D66N mutant using quick change site directed mutagenesis. Harkewal Singh 09/01/2010

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1 Engineering D66N mutant using quick change site directed mutagenesis Harkewal Singh 09/01/2010 1

2 1- What is quick change site directed mutagenesis? 2- An overview of the kit contents. 3- A brief information on the working conditions. 4- A general outline of the process. 5- Troubleshooting. 6- Examples from D66N mutation. 2

3 Quick change site directed mutagenesis is the process of introducing a site specific* mutation into double stranded plasmid. does not require any specialized vectors, unique restriction sites, or multiple transformation. site specific* D66N mutation [ V V A D L D E T ]- - [ V V A D L N E T ]- 3

4 Overview of the kit contents 1-10x reaction buffer - This is for the maximum/optimal activity of Pfu DNA polymerase. Contains ~ 25mM MgCl2,Triton X-100,100 mm Tris-HCl, 500mM KCl, ph Pfu Turbo DNA polymerase - It is a thermostable enzyme isolated from Pyrococcus furiosus. It catalyzes the plolymerization of nucleotides into double stranded DNA in the 5-3 direction. 3- Dpn1 restriction enzyme - recognizes methylated DNA bases. 4- dntp mix - deoxy ribose nucleotide triphosphate(dntp) 5- XL10 - Gold Ultracompetent cells 4

5 Overview of the working conditions 1- Thaw dntp only once, so prepare single use aliquots and store them at -20 C. 2- Thawing MUST be done at ice in case you have more than single use aliquots. 3- All the procedures which involve kit contents MUST be done on ice. 4-Take Pfu DNA polymerase LAST out of the kit and put it back as soon as you are done. 5- Water must be autoclaved and free of any debris or fungal/bacterial contaminants. 6- Use sterile tips for pipetting out any of the kit contents. 7- Always freeze XL10-Gold cells at -80 C. 8- Finally, use thin walled autoclaved PCR tubes for the experiment. 5

6 General outline of the procedure 6

7 7

8 Troubleshooting 1- Low transformation efficiency or low colony number - Make sure that DNA template is of good quality by running on a gel. Also check the quantity by measuring the concentration of your template DNA. 2- No colonies at all - Do an ethanol precipitation* of your PCR product and re-transform using XL10- Gold ultracompetent cells. 3- Low mutagenesis efficiency could be due the insufficient digest of parental DNA with dpn1 so increase the digest time from 1 hour to little longer e.g hours at 37 C. 4- Also AVOID multiple freeze thaw cycles of dntp mix, this could lead to low mutagenesis efficiency. 5- False positives - meaning you have great colonies but no mutants. Make sure your primers are correct. or wrong extension temperature. 8

9 Ethanol Precipitation 1. Add 1/10 volume of Sodium Acetate (3 M, ph 5.2). 2. Add times volume of 95% ethanol. 3. Incubate at either -20 C or -80 C overnight. 4. Centrifuge at > 14,000 x g for minutes at 4 C. 5. Discard supernatant being careful not to throw out DNA pellet which may or may not be visible. 6. Rinse with 70% Ethanol. 7. Centrifuge again for minutes at > 14,000 x g. 8. Discard supernatant and dissolve pellet in 10mM Tris ph Now you can use this DNA (~5ul) and retransform using XL10 Gold ultracompetent cells. 9

10 Engineering D66N mutant of P4 enzyme Requirements 1- Template DNA( wild type plasmid cloned into pet20b using nco1/xho1 restriction site. ~ 100 ng/ul ) 2- Primers (forward and reverse ) in this case D66N forward* and D66N reverse*. (make the concentration ~ 500 ng/ul) 3-10 X reaction buffer as supplied with the kit.(thaw on ICE) 4- dntp (thaw on ICE). 5- QuickSolution reagent. 6- Autoclaved Water. 7- Autoclaved thin walled tubes. 8- Autoclaved pipette tips 9 - Pfu DNA polymerase as supplied with the kit(at last) 10

Primer design guidelines 1- Both primers MUST have the same mutation. 2- Primers length should be between 25-45 bases. 3- Tm should be 75 C or greater.

11 Primer design guidelines 1- Both primers MUST have the same mutation. 2- Primers length should be between bases. 3- Tm should be 75 C or greater. 4- The desired mutation should be in the middle of the primer. 5- In general its good to have the GC content of a primer around %. 6- The primer should end in G or C. 7- Calculate Tm of the primer using either some webserver or this formula N - primer length % mismatch - change in the base used to make mutation. 8- Finally, make up your primer volume by adding either autoclaved10mm Tris ph 8.0 or autoclaved water, the former is recommended. 11

12 Wild type sequence Asp Leu Asp 5 GCG GTT GTG GCT GAT TTA GAT GAA ACT ATG TTA GAC AAC 3 Forward primer design Asp Leu Asn 5 GCG GTT GTG GCT GAT TTA AAT GAA ACT ATG TTA GAC AAC 3 fwd Reverse primer design Complement of sense strand 3 CGC CAA CAC CGA CTA AAT TTA CTT TGA TAC AAT CTG TTG 5 Final reverse primer 5 GTT GTC TAA CAT AGT TTC ATT TAA ATC AGC CAC AAC CGC 3 rev 12

13 wild type sequence CCA GTT GTA TTC ATG GAC ATT GAC GAA ACT GTG CTG CAG 13

14 wild type sequence CCA GTT GTA TTC ATG GAC ATT GAC GAA ACT GTG CTG CAG forward CCA GTT GTA TTC ATG GAC ATT AAC GAA ACT GTG CTG CAG 14

15 wild type sequence CCA GTT GTA TTC ATG GAC ATT GAC GAA ACT GTG CTG CAG forward CCA GTT GTA TTC ATG GAC ATT AAC GAA ACT GTG CTG CAG reverse CTG CAG CAC AGT TTC GTT AAT GTC CAT GAA TAC AAC TGG 15

16 PCR reaction setup for D66N mutation Component Volume 1 10 X reaction buffer 5 ul 2 DNA template (P4 wild type DNA) 0.25 ul or 10 ng 3 D66N forward primer 0.25 ul or 125 ng 4 D66N reverse primer 0.25 ul or 125 ng 5 dntp mix 1ul 6 QuickSolution reagent 3ul 7 Sterile water ul (mix well after this) 8 Pfu DNA polymerase 1ul(mix again) 9 Total 50 ul 16

17 Segment Cycles Temperature Time C 1 minute C 60 C 68 C 50 secs 50 secs 2 min/kb of plasmid length * C 7 minutes 4 4 C hold as little as possible * pet20b is ~ 5kb and P4 gene is ~ 1 kb so total length to amplify = ~6 kb hence I used 14 minutes for elongation time. Add 1ul dpn1 to the PCR tube when the reaction is complete, mix well, spin for two minutes at ~ rpm and incubate the PCR tube at 37 C between hours 17

18 1- Transform the PCR product (1-2 ul) in ul of XL10 Gold cells as usual. 2- Plate on LBagar supplemented with appropriate antibiotic. (Amp50 in my case) 3- Next day (Day 2), pick three - four single colonies and grow in LB broth supplemented with Amp50 or your AB. 4- Do plasmid prep and before submitting all your potential constructs for sequencing verify to check the success your PCR. 5- wild type P4 was cloned using nco1/xho1 into pet20b, so I digested the plasmids using nco1 and xho1 enzymes at 37 C and then ran % agarose gel. 6- The gel showed that I had a band around 1 Kb which suggest that the PCR was a success. 7- Now submit one or maximum two clones for DNA sequencing. 18

19 gene of interest 19

20 DNA sequencing result MAMGSHQMKSEEHANMQLQQQAVLGLNWMQDSGEYKALAYQAYNAAKVAFDHAK VAKGKKKAVVADLNETMLDNSPYAGWQVQNNKPFDGKDWTRWVDARQSRAVPGA VEFNNYVNSHNGKVFYVTNRKDSTEKSGTIDDMKRLGFNGVEESAFYLKKDKSA KAARFAEIEKQGYEIVLYVGDNLDDFGNTVYGKLNADRRAFVDQNQGKFGKTFI MLPNANYGGWEGGLAEGYFKKDTQGQIKARLDAVQAWDGKLEHHHHHH 20

Add 5µl of 3N NaOH to DNA sample (final concentration 0.3N NaOH).

Bisulfite Treatment of DNA Dilute DNA sample to 2µg DNA in 50µl ddh 2 O. Add 5µl of 3N NaOH to DNA sample (final concentration 0.3N NaOH). Incubate in a 37ºC water bath for 30 minutes. To 55µl samples