Mouse MSP primers - (Nov/22/2005 )

Hi,I am very glad to see posts about methylation protocol here.
I have already tried in this aspect for a long time.I bought a kit about p16 ,modification and methylation.And I tried with Human samples.I have gotten very good results,and the bands were very clear.Then I tried with mouse samples,extracted DNA from mouse spleen,modificated and amplified with the primer designed like pcrman mentioned.But no bands appeared.And I tried with more primers like p15,pten,etc,still no band.
Could you please tell me what is my problem?
Comparing with the kit for human(no kit for mouse),there is only difference with primers.I wonder if I should modificate the primer designed with methprimer?
I am a new postdoc here and I just can not do this well.I am very hopeful for your help.Thanks!

how homolgous is p16, pten and the like between human and mouse? you may need to design specifc primers for mouse p16, pten etc as the sequence may be different enough for the primers for human samples not to work on mouse.

I would suggest you have a go at designing your primers by yourself. Methprimer is a convienent way of picking primers but i have to say it's not picking optimal primers for bisulfite pcr and sequencing.

N

Thank you so much for your help.
But actually I design specifc primers from the sequence of mouse p16, pten with Methprimer. I t just do not work.

Could you please tell me how I can design primers by myself?Have you tried about that?Maybe the software have not given me the optimal primers. Thanks!Lisa

I did the mouse PTEN by using Methprimer and my MSP works fine.

Thank you so much for your help.
But actually I design specifc primers from the sequence of mouse p16, pten with Methprimer. I t just do not work.

Could you please tell me how I can design primers by myself?Have you tried about that?Maybe the software have not given me the optimal primers. Thanks!Lisa

Could you please give me some guidance?
First I designed the primers,my primer as:
Primer Start Size Tm GC% 'C's Sequence
Left M primer 127 25 59.85 80.00 12 TTAGGTTTGTTCGTTTCGTTTATTC
Right M primer 233 24 59.68 58.33 4 ACCCTATCGAACCTTACATATTCG
Product size: 107, Tm: 66.9
Left U primer 129 25 57.41 80.00 11 AGGTTTGTTTGTTTTGTTTATTTGT
Right U primer 232 25 57.74 60.00 5 CCCTATCAAACCTTACATATTCAAA
Product size: 104, Tm: 67.1
I used mouse spleen to extract DNA,the purity(ratio) of DNA is about 1.800.and I have midificated DNA ,the PCR protocol is :95-8min(hot-start),95-45sec,55-45sec,72-1min,35cycle.
The result is no band at all.Could you see what is my problem?what I should try again?
Thanks!
Lisa

Lisa,

can you also upload the sequenes as well?

if you performed conventional bisulfite PCR, you may need to perform two rounds of PCR as you have very little starting DNA.

You may need to amplify the region your U and M primers with generic converted primers and then use your U and M primers in a second round of PCR.


There is a new technique called hairpin bisulfite PCR that may be of interest to you. Pubmed this and you will get the reference.

Cheers

Nick

Hi,nick,
I forgot my password so I changed a name to log in here.
I designed the primer from the sequence of ENSMUST00000013807.
Do hairpin bisulfite PCR like nested PCR?I want to try this and see some reference.
Thank you so much for your help.You are really a pro in this field.
Lisa

Hi Lisa,

I had a look at ENSEMBL, and I am not too familiar with it as I use Genome Browser all the time these days. From genome browser, I gather you would like to look at the methylation status of CpG island of pten, looking within genome browser, this is slightly upstream of the pten promoter (see attachement), so I would direct my efforts on this. I think from your primer sets, the primers are directed to sites after the start of the transcript. I tried methprimer but it is not behaving, maybe you should try this sequence I have also attached which is the CpG island with 500bp of flanking sequence from Genome Browser also.

I have also attached the reference with regard to hairpin bisulfite, it's actually headloop bisulfite, but i remember it as hairpin :-). The paper can be accessed through pubmed for free so I guess it wouldn't be much of a problem to post it here (save you all time going to pubmed)

see how you go with this lisa.

Nick

here is the text file:

>mm6_dna range=chr19:32081537-32085084 5'pad=0 3'pad=0 revComp=FALSE strand=? repeatMasking=none
tatgaaattttccttctcctgtactctaaacccccagctagcctttccta
gacacctactcgcaattattgcaaatccataactgactactatcctccgg
atttctaaaatgatccagtgtttcagcttaggtctcaactcagagatact
ttagggctcagattggcatcctgagaattaagtcccctgggaaaagaaca
ataaggaagaaaactctacctacattggagttgatgtcattttttttttc
cctccaagctcaaggtgatcgcttgctttgtggctggttggtgggggagg
aggggctgtacgctagttatcagcatttctgaaccagctctctcaaccgc
gacaggtcagccaatcccggcagtaagcttttacttgacaggtttgttct
gggctgacagccattgactaggtgctcagataagtcacttggctgagtct
acggtaggtggggcgcgctcaccagttcaggggcagtgactggaagtttg
ttgcaacatcggtaagcctaaccagccagcagcaacaggagatacccttt
tgccccgcgagtacagatctagaaagggttcacctcattaagcgaaggag
atgcgtcaatcccccccacccccgccccgcgcctccccctagggcccggc
ctcttctcccacggttgggaacgcgcggtgtgggcagatccagaacagga
gtctcgtgtcccggccttctggctagctctatgggttacaagcgaaaggg
aggaacagcttggggactctccgcgtcagcgtgcacaaaccggcggcggc
cagcagagaggggtggcgggggcacgtgcttggatgtggctgcttgtgta
accagctccccaggcgctcggccccgacagcgctcctgcggacggctcgt
ggatgctattctctgctccgatccggcaagagaggggtccagcagaccac
acgggagaaggaggcgggggcgatcacctaatagagcagaggggaccaag
ctcctgccccaggagcacacagataggggaatgggaatttggaaagttcc
ccaactaggaccacacgtgacctcctcctgaaagtagttccgaccgcggc
tcatgtatccttccacctcgcctttgagccctcccaggcctgctcgcccc
gcccactcgctggctgcagcttccgaacgtcccatactccacacccgggc
tcagtaaccgggtcctcgaacatgcaaggtccgacagggtcagaacctgg
ccatcgcgatccaattctgccgggttttcatagcggccacgaagtgggga
ttgggggtgggggcttagctctttgaagactgagcttggctgtgatccgg
tagacccaccgctgcggggagctgcgggtctcatcaccgggcggtggagg
ggtgtgtgtgaggtgcactctattcacggagacccactttgtccaaccag
gggtgtcctttgggccctggaaactcaggggagatgtgaatgtacacgcc
ccgtatgcacaatcatcatgcttggctgggagcgttcatctttcgggcaa
atgaacccagctgcctgggaagcaagaggcggggcagggaaccggagccc
gatgaggtgacccacgcgggagacacaataggggttgttctttgtgcaaa
gactgacaccttgaggacaccgtgagggggagaggtgtgttatctaggta
aagactgtcgccgacaaatcctagcgaagcactgcaatctgaccacagcg
cagggcagggaatgaaagccgttccgaagaaacgcagggacagacgcagg
aaggataatcctgcccctgaggctcccggagcaccgaccaaggcggtcag
ctagtgcgatccacctgtgagcggtcagcgattgtgctcagcgcaccctc
actcggccccagcctgttgtacctttgccgggtctctctgcgctgaggcc
aaagccggcgtagctccgggagcgagccgcggacacactgggcatgctcc
gcggcgttccccgcccctgtcccttccgacgccccgccccgccccgcccc
gtccccggctcagcgcccgcctcccgcccgcctcccgcctcccctccggc
tttccgaggcgccctgctctcccggcggggcggcggagggggcgggctgg
ccggcgcacggtgatgtggcgggactctttgtgcactgcggcaggatacg
cgcttgggcgtcgggacgcggctgcgctcagctctctcctctcggaagct
gcagccatgatggaagtttgagagttgagccgctgtgaggccaggcccgg
cgcaggcgagggagatgagagacggcggcggccacggcccagagcccctc
tcagcgcctgtgagcagccgcgggggcagcgccctcggggagccggccgg
gcggcggcggcggcagcggcggcgggcctcgcctcctcgtcgtctgttct
aaccgggcagcttctgagcagcttcggagagagacggtggaagaagccgt
gggctcgagcgggagccggcgcaggctcggcggctgcacctcccgctcct
ggagcgggggggagaagcggcggcggcggccgcggctccggggagggggt
cggagtcgcctgtcaccattgccagggctgggaacgccggagagttgctc
tctccccttctcctgcctccaacacggcggcggcggcggcggcacgtcca
gggacccgggccggtgttaagcctcccgtccgccgccgccgcaccccccc
tggcccgggctccggaggccgccggaggaggcagccgctgcgaggattat
ccgtcttctccccattccgctgcctcggctgccaggcctctggctgctga
ggagaagcaggcccagtctctgcaaccatccagcagccgccgcagcagcc
attacccggctgcggtccagggccaagcggcagcagagcgaggggcatca
gcgaccgccaagtccagagccatttccatcctgcagaagaagcctcgcca
ccagcagcttctgccatctctctcctcctttttcttcagccacaggctcc
cagacatgacagccatcatcaaagagatcgttagcagaaacaaaaggaga
tatcaagaggatggattcgacttagacttgacctgtatccatttctgcgg
ctgttcctctttgcttttctgtcactctgataacgtgggagtagacggat
gcgaaaaatttctgtagttggggtgactataacgtttaattctgggcgca
tttctagatcgtgcatattgtgtctcttccagtgtattcaacctagggag
tgttcggctagacggaactcatgcctccttgcaagtgtcaaggcacggat
tgttttcttgtcgagctctgtggtctcttcttaaaatctattgtccggta
atacagagtactgtacactggattagcgagctcgtcaatccagccttcta
aatgaactaaaaaaaaaaaaaaaaatagacgctttgggttgtgcatattt
cgattagatcgtgacttgggccctagatctagggtgtagatgagatta

Thank you,Nick!you are right.
Before I used ENSEMBL to search sequence.But Recently I found it was difficult to repeat it again.Maybe the website has always been updated and some sequence are not so precise.

Could you please have a look at my another primer from P16 promoter?
ENSMUST00000030237
P16 Primer Start Size Tm GC% 'C's Sequence
Left M primer 363 25 59.02 40.00 4 AAATCGAAAATAAATAACGTTTTCG
Right M primer 544 23 59.62 60.87 4 TAAACCCTTAACGATACGCTACG
Product size: 182, Tm: 72.2
Left U primer 364 25 55.14 44.00 4 AATTGAAAATAAATAATGTTTTTGG
Right U primer 544 25 53.52 60.00 4 TAAACCCTTAACAATACACTACACT
Product size: 181, Tm: 69.5
I can not amplify it too.
Thank you for all your help!
Lisa