I have primers from Integrated DNA Technologies in lyophilized form. The amount of Oligo is 25.5 nMoles = 0.16 mg. Now how should I dissolve it? Which solvent should I use? And how much of it?
Assalam-o-Alaikum Mr Huq
Please see the calculations below
Multiply (25.5 nMole ) with 10 = 25.2 x 10= 255 ul.
Add 255 ul of molecular biology grade water to the vial of oligo.
This will be your stock solution of 100 µM (micro-molar).
Best Regards
You can dissolve primers in: (1) Autoclaved milli-Q grade water: Add worm water (65 temp), and incubate at 65 temp in dry/water bath for ~10 min with the occasional mixing by vortex. (2) 10 mM of TE (pH-8) by vortex and proper mixing, put tubes overnight at 4 temp will enhance primer dissolution.
Generally people make 100 pmol main stock, and working may be 10 or 20 pmol. For 100 pmol main stock you can add 255 µl either sterile mill-Q water or 10 mM TE (pH-8).
Spin the primer tubes at 10k for 15 mins. Dilute to 1nanomole concentration. Ur primer concentration is 25.5 nM. Add 25.5 microlitre TE ph 8. Incubate in ice for 30 mins. Vortex and maximum spin for 10 mins at 4°c. Dilute 20x and then primer is reay to use.
Assalam-o-Alaikum Mr Huq
Please see the calculations below
Multiply (25.5 nMole ) with 10 = 25.2 x 10= 255 ul.
Add 255 ul of molecular biology grade water to the vial of oligo.
This will be your stock solution of 100 µM (micro-molar).
Best Regards
E.g. 200 umol/L = 200 nmol/ml = 200 pmol/ul
Your oligo is 25,5 nmol = 25,5 nmol/ml = 25,5 pmol/ul
If in a 1 ml you have 200 nmol, in a 25,5 nmol how many ml?
1ml : 200 nmol= x : 25,5 nmol
x= 25,5 nmol x 1ml / 200 nmol= 0,1275 ml (127,5 µl).
127,5 ul is the volume of TE or milli-Q to add in your lyophilized tube in order to abtain a 200 pmol/ul = 200 uM stock solution.
You can dilute 1:10 to obtain a work solution of 20 pmol/ul (10 ul of stock sol + 90 TE)
for PCR see http://www.qiagen.com/knowledge-and-support/spotlight/protocols-and-applications-guide/pcr/ or https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase
for QuikChange see for instance http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0CEUQFjAC&url=http%3A%2F%2Fsevierlab.vet.cornell.edu%2Fresources%2FStratagene-QuikchangeManual.pdf&ei=fH0xU4qUGsyFtQaT5oDoCg&usg=AFQjCNF4B34fUF6nG_m_91opdncMrE3WJA&bvm=bv.63587204,d.Yms
@Ditte: Thank you for "Take a look at the papers that came with the primers - maybe they hold more information than you think :)" - sometimes it seems to be easier to load questions onto other people than to try to retrieve the information by some effort...
Thank you all for your advises and suggestion. @Ulrich: I do have a data sheet and nothing mentioned there how to dissolve the primers. But later on my friend told me to look at their website where they have a resuspension calculator to calculate the volume. Now my point is how a person would know that you have look at their website if it is not mentioned in the sheet or vial label?
Materials
‣Molecular Grade H2O
‣Primers (dry)
‣Sterile Microcentrifuge Tubes
Method
Primers are often shipped and received in a lyophilized state. First create a master 100
× stock (for each primer and then dilute it to a 10× working stock.
This reduces the number of freeze/thaw cycles that the master primer stock goes through
and reduces the chances of contaminating the primary source for the primer.
Spin Down Tubes
Primers should always be spun down before opening the tube for the first time. The pellet can often come dislodged during shipping and may be in the cap!
Master stock, 100 µM
100 µM = X nmoles lyophilized primer + (X × 10 µl molecular grade H2O)
To determine the amount of H2O to add to the lyophilized primer simply multiply the number of nmol of primer in the tube by 10 and that will be the amount of H2O to add to make a 100 µM primer stock.
For example, if there are 38.2 nmol of primer then by adding 382 µl of H2O, a 100 µM primer stock is created.
The original primer tubes are often used for this 100 µM stock.
Master stock primers newly suspended in H2O should be allowed to sit at room temperature for 10 minutes before they are used for working stock dilutions. Mix well before making working stock dilutions.
Working stock, 10 µM
Dilute the primer master stock in a sterile microcentrifuge tube 1:10 with molecular grade H2O.
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