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PCR: Cloning Primers


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#1 Fluffy

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Posted 19 May 2011 - 11:09 AM

Hi Everyone,
I have to design forward and reverse primers for the full sequence of my two genes (ubiquitin conjugating enzyme genes). Primers have to include 5' restriction enzymes. My restriction enzymes are NdeI (forward primer) and XhoI (reverse primer). I was told that since I am designing primers to amplify full sequence of each gene, the start and stop codons in each gene sequence have to be considered. Primer length is 18-25 ntds preferably, GC content below 50%, and Tm range is 58-60C. I am not good at using softwares for primer design, so I would appreciate the help of anyone able to use primer design tools. The gene sequence for my two genes are as follows:

Gene 1 sequence ID 388206
1 ctcttcttcc atttctttca aaattaaagt attgttactc tgctattggc tcaaaacctc
61 tgcaatctcc gtctccttca atttcaactc aagcaaatcc acctctttca ctagtttcat
121 cactttcaga tcagggtttg gagttgaagg tacggggggc taattgatgg cgtcgaagag
181 gatattgaag gagctcaagg atctgcagaa ggatcccccc acatcatgca gtgctggtcc
241 agtggcagag gatatgttcc attggcaagc aacaatcatg gggcctaccg atagccctta
301 tgctggaggt gtatttttgg tttcaattca tttccctcca gattatcctt ttaagcctcc
361 aaaggttgcc ttcagaacta aggttttcca tcccaacatc aacagcaatg gaagtatttg
421 tctggatatt cttaaggagc agtggagtcc agcattaacc atatccaagg tcctgctgtc
481 catctgctct ctgttgacag acccaaaccc agatgatcct cttgtacctg aaattgctca
541 catgtacaag actgacaggg ccaaatacga aaccactgct cgtagctgga ctcagaaata
601 tgcaatggga tgatgcgcaa aatgtctcca ggcatgtctg ggactttgta acagcaatgt
661 cttatgtgct tggggtgaat gaataaattc cgtgaaagaa cttagttact tcttaatctc
721 ccttcatgag ggttgttaag ggaacagctg ttttcaattt gtgaatattt atttgatgac
781 tagtaaggga gaaactgcaa tgtaattcta ctttgtttgc cagtt

Gene 2 Sequence ID 886678
1 gcacgagggc gacttttgca taaaccaaaa ttagaatcaa attggaagag agaaaaaaaa
61 tggtggactt ggctagggtt caaaaggagc tccatgaatg caacagagat gttcaggttt
121 ctggaattaa tgttaccctt aaaggtgaca gtctcactca cttgattggt acaatccctg
181 gtcctgttgg tactccttac gaaggcggta ctttcaagat cgatatcact cttactgatg
241 gctacccatt tgagcctcca aaaatgaaat tcgccacaaa agtttggcat cccaacataa
301 gtagtcaaag tggagcaata tgcctagaca tcctgaagga ccagtggagc ccagcactaa
361 ctctcaagac agctctcctt tctatacaag cattactttc tgctcctgaa cctgatgatc
421 cacaagatgc agttgttgca cagcagtatc ttagagaaca tcagaccttt gtcggcacag
481 ctcgttactg gactgagact tttgcaaaaa catccacact tgctgcagac gacaagatac
541 aaaagcttgt ggaaatgggc tttcctgaag ctcaagtgag gagtactttg gaagcaaatg
601 gttgggatga aaacatggct cttgaaaagc tgttgtccag ctaaaaccct tctactgcaa
661 ctcatatttt gataagacaa ttatatcctt ccagcaaaag ctgatgacta gaatagagtc
721 actcggttat actgttgctt ggcaatcttg tttctgtctc ctttatggtt tgctgttgac
781 atctcttcat atcctgtgaa gattctgatg ttatttttac aatatcaagc aaattgcata
841 tgaatcatgg ggaggaagtg gactttccgg ggtgaaaaaa aaaaaaaaaa aaaaaaaaaa
901 aaaaaaaaaa aaaaaaaaaa aaa

Thanks Everyone :D:D

#2 bob1

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Posted 19 May 2011 - 05:15 PM

First of all you need to identify where the start and stop codons are in those sequences... unless you want to clone the UTRs also.

If you want to clone the full sequence excluding UTRs, then you have no choice about where to place the primers, other than length... so your specific primers are the start 18-25 bp and the final 18-25 bp (reverse complemented) of your gene sequences. YOu can calculate the Tm by using the following formula: Tm= 4*(number of G and C) + 2*(number of A and T). You can adjust the length of the primers to compensate for differences in the sequence giving different annealing temperatures.

The annealing temperature you will use in the PCR is calculated based on the specific component only, ignoring the bits added as these don't play a part in the PCR initially.

Restriction sites and other add-ons go on the 5' end of the primer always.

You will probably want to add a kozak sequence (GCCACCATG, where the ATG is the start codon of your gene) to the 5' end if you are planning on expressing these in eukaryotic cells.

So your forward primer will look something like:
[Nde1site][kozaksequence][18-20bp of codingsequence]

Reverse will look like:
[Xho1site][reversecomplement of 18-20 bp of codingsequence]

#3 Fluffy

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Posted 20 May 2011 - 10:41 AM

Hey Bob1
Thanks for your help!
Greatly appreciated :D

#4 pmggms2

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Posted 23 May 2013 - 11:26 AM

What is a kozak sequence and do you need to add them when cloning?

#5 bob1

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Posted 23 May 2013 - 12:34 PM

What is a kozak sequence and do you need to add them when cloning?

here

#6 phage434

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Posted 23 May 2013 - 02:57 PM

You need some additional 5' bases outside of the restriction enzyme region. I'd recommend around 6, for most enzymes. It doesn't matter what they are as long as they don't result in hairpins or other secondary structure.

#7 notjustalabtech

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Posted 09 June 2013 - 10:39 AM

You need some additional 5' bases outside of the restriction enzyme region. I'd recommend around 6, for most enzymes. It doesn't matter what they are as long as they don't result in hairpins or other secondary structure.



Yes, usually TTTTTT+restrictionsite+primer has worked for me. Also make sure that when calculating the proper Tm for each primer, you don't include the TTTTT tail or the restriction site, since they aren't technically annealing onto the gene.

#8 Euterpa12

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Posted 03 July 2013 - 02:13 AM

When ordering my primers, I forgot to add additional bases in front of the restriction site. Will my digestion work like this at all, or do I have to order new primers?

#9 phage434

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Posted 03 July 2013 - 06:09 AM

This probably depends a lot on the enzyme, but my guess would be that it probably won't work. You could try lowering the temperature of digestion, which might make things work a bit better.




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