I have a question in choosing primers for sequencing. Which one is preferable: using only M13 (-21) or sp6 and T7 forward and reverse primers, respectively? As I don't know the orientation of my insert, I'm likely to amplify the complementary to my insert than the insert itself if I use only M13 (-21) which could result in taking complementary sequence for annotation. How can I solve this limitations in using single (only forward) primer (M13-21)? I think I have to use sp6 and T7 as either of the two surely amplify my insert as the amplification occurs in both orientations. My vector was pGEM-T easy.
I am using pGEM-T easy and I use T7 and sp6 to sequence my fragments and they are working perfectly.
txs yordan!!
One more thing, what is the conc. of both primers (sp6 & t7) that you are using?
Hi, Geleta!For the sequencing I am using 3.2pmol either T7 or sp6 primers in 20µl reaction and also I use CEQ DTCS Quick start kit from beckman coulter. the annealing T for the reaction is 50 deg. The protocol is according to the kit. I don't know if you are going to do the sequencing by yourself or not but if you need anything just tell me.
I also used (and very much like it) T7 and SP6 to sequence my insert. I would do both directions (meaning use of both T7 and SP6 from two directions). One direction (T7) will cover a good reading on one end of your insert sequence, the other direction (SP6) will cover a good reading on the other end of your insert sequence (remember that machine normally gets some 'noise' sequence reading close to the primer.) Then I will assemble/edit these resulting sequences together with a DNA Sequence Analysis software (such as SequencherTM).
Dear Yordan, I'm using ABI PRISM BigDye sequencer with an assistance from a technician on loading the samples. You mean either sp6 or t7 are used or both are used in a SINGLE REACTION (as forward and reverse primers) so that you will find out (during annotation) which of the primers actually amplified your insert that match the orientation of either primers? OR either of the primers (sp6 or t7) is used in a reaction? I think it's better to use both in A REACTION, right? That's so because one of the primer is expected to amplify the complementary strand rather than the the actual insert template sequence. Thankyou all guys am enjoying...
Hi, in my sequencing kit you are using only one sequencing primer either T7 or sp6. If you wish to sequence the both DNA strands you have to set 2 reactions with only one primer each. This is for beckman coilter kit , the one i mention above. Perhaps in your system you have to use 2 but in my case only one. Good luck!
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