Add+PolyT+near+Lac+Promoter+8-28-12

8-28-12

Plan to add poly T near Lac promoter with new primers (the old primers were incorrect).

Updated Oligos for adding polyT APT (for add polyT) GCTGGTTTCGCTACCGTGGCCCTTTTTTTTTTTTGGCCCAGGCGGCCGAGCTCGCCATG RPTFC (for reverse polyT fragment corrected) AAAAAAAAAGCGGCCGCCTAGTAGAACCGTAG APT2 (for add polyT 2 which corrects any mistakes with the 1st long primer APT) GTTTCGCTACCGGGCCCTTT RPTFC2 AAAAAAAAAGCGGCCGCAGAACC SAPTFC (for sequence add polyT forward corrected) AGCGCCCAATACGCAAACCG SAPTRC (for sequence add polyT reverse corrected) TGTGGAATTGTGAGCGGATAACAATTTC

I also have two backup primers in case these ones do not work. RPTFCB (for reverse poly T fragment corrected backup) GCTAAAAAAAAAAAAAGCGGCCGCCTAGTAGAACCGTAG RPTFC2B (for reverse poly T fragment corrected 2 backup) GCTAAAAAAAAAAAAAGCGGCCGCAGAACC

To use these primers, I need to cut the plasmid that has already been modified with SfiI and NotI (MN 7-21-12 (MN is for modified NotI)). The fragment needs to be amplified with APT and RPTFC primers. Then I need to reamplify with APT2 and RPTFC2 primers. Then I need to perform the In-Fusion reaction with the plasmid backbone. This plasmid will need to be transformed into some bacteria. Then this plasmid will need to be sequenced with SAPTFC and SAPTRC.

The 1st step is to cut MN 7-21-12 with SfiI and NotI, and then gel extract.

Approximate size of expected fragment is about 1.6 kb Plasmid is about 5 kb Plasmid backbone is about 3.4 kb

Actually I found the already cut and gel extract fragment that I had from before (pComb fragment 7-6-12 gel extracted). I will PCR this fragment using the Primestar PCR system (see Primestar Max DNA Polymerase). PCR will be performed with APT and RPTFC primers. The PCR product will be gel extracted.

Items: pComb fragment gel extracted 8-28-12 (this fragment was amplified with the APT and RPTFC primers but not the 2nd round of correction primers (APT2 and RPTFC2) pComb fragment gel extracted 1 8-29-12 (this fragment was amplified with APT2 and RPTFC2 and is 1 of 3 fragments gel extracted since I wasn't completely sure which one was correct (see notebook for gel image). This fragment will be used for an In-Fusion reaction) pComb fragment gel extracted 2 8-29-12 (this fragment was amplified with APT2 and RPTFC2 and is 1 of 3 fragments gel extracted since I wasn't completely sure which one was correct (see notebook for gel image). This fragment will be used for an In-Fusion reaction) pComb fragment gel extracted 3 8-29-12 (this fragment was amplified with APT2 and RPTFC2 and is 1 of 3 fragments gel extracted since I wasn't completely sure which one was correct (see notebook for gel image). This fragment will be used for an In-Fusion reaction)

The above 3 fragments were performed with an In-fusion reaction with the pComb plasmid 7-6-12 gel extracted. These plasmids were then electroporated into DH10B cells. Different colonies from each of the three plasmids were then sent for sequencing to see if the poly T modification was truly added near the lac promoter.

Items mpComb (for modified pComb3xSS) 1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 2.4, 3.1, 3.2 (10 different samples) 9-5-12 stored at 10-3-11 -20 C

9-15-12 Sequencing lab e-mailed me back with results on 9-7-12 ( https://mail.google.com/mail/u/0/?ui=2&shva=1#label/Career/139a280d2bc1092e)

Sequences stored here "C:\kurt\storage\CIM Research Folder\DR\2012\9-18-12\pComb Mod Sequences 9-7-12 Kurt"

Now I need to see how these sequences align with my expected modified pComb sequences. After looking at the sequences, I see that not one of them sequenced well so I really can't do anything with these sequences. I think one possible reason for these poor results could be that I let the plate overgrow. Therefore, there were colonies growing on the plate which did not contain the modified pComb plasmid. I think I will try to resequence just one of the colonies with both the SAPTF and SAPTR primers as well as the other sequencing primers (SANotI and SRNotI). I will then start trying to make this modification starting from the very beginning again. When I restart, I will perform the PCRs with a higher fidelity polymerase like Advantage 2 instead of PrimeStar. see Add PolyT near Lac Promoter 9-18-12

I sent mpComb 1.1 9-5-12 for sequencing one more time with the SAPTF, SAPTR, SANotI, and SRNotI primers.