Add+PolyT+near+Lac+Promoter+070612

The polyT site will be added by cutting with SfiI and NotI. The fragment needs to be amplified with APT and RPTF primers. Then I need to reamplify with APT2 and RPTF2 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 SAPTF and SAPTR.
 * Plan**

I will use the Primestar Max DNA Polymerase.

7-6-12 First I need to cut the vector with SfiI and NotI. see Restriction Digest Protocol Approximate size of expected fragment is about 1.6 kb Plasmid is about 5 kb Plasmid backbone is about 3.4 kb

The restriction digest worked just as expected. Items
 * pComb backbone 7-6-12 KW gel extracted
 * pComb fragment 7-6-12 KW gel extracted

7-7-12 Performed PCR and gel extracted 7-8-12 Performed PCR with shorter primers and gel extracted 7-9-12 Performed In-Fusion reaction

Sequencing Results L:\storage\CIM Research Folder\DR\2012\7-16-12

Sequencing Results (2nd try) L:\storage\CIM Research Folder\DR\2012\7-18-12\pcomb sequence analysis

Some Notes on analysis > L:\storage\CIM Research Folder\DR\2012\7-19-12\1507 > Here's a screenshot showing that the forward and reverse sequences match well without reverse complementing (these are the first sequencing results from the last experiment to modify the NotI region) > "L:\storage\CIM Research Folder\DR\2012\7-19-12\1507\1515.png" > Now I will try taking the reverse complement and comparing the two. > clustal input: > "L:\storage\CIM Research Folder\DR\2012\7-19-12\1507\input 1522.txt" > clustal output > "L:\storage\CIM Research Folder\DR\2012\7-19-12\1507\1526.png" > When I take the reverse complement the sequences do not align well. > "L:\storage\CIM Research Folder\DR\2012\7-19-12\1507\1526.png" > This was very strange. Now I know what the problem was though after some discussion with Andrey and Felicia. I was obtaining my sequences from a dissolved contig in sequencher. I thought that when I dissolve a contig that the sequence would be in the exact same format that it came in from the sequencing lab. However, once a sequence has been included into a contig in sequencher, the format of the sequence (forward or reverse complement) is maintained, which I did not expect. Now that I know this, I can test Clustal again. From what I understand so far, it looks like Clustal requires proper reverse complemented sequences for good alignments and the program will not do this on it's own. > 1633 Now everything makes sense. Clustal does require sequences in the proper format (forward or reverse complement from a reverse primer sequence). When I reverse complement the original sequence from the sequencing lab, everything aligns properly. > Now that I know that Clustal requires proper orientation sequences, I can go back and look at my sequencing analysis for the 7-16-12 sequencing results.
 * I realized that when I first looked at these results, I was not reversing complementing the reverse sequences and maybe I should have. I will look at this more.

These results were not ideal. This may be due to the fact that I did not have a full 150 ng of plasmid. The in-fusion 1 reaction only had 85.5 ng in the tube, and the in-fusion 2 reaction only had 135 ng in the tube.

Sequencing Results (3rd try) 7-22-12 (Sun)(Sequencing lab will get it Mon morning 7-23-12) Miniprepped more plasmid

Sequenced again. However, this time I used various concentrations of primer (1/2X, 1X, and 2X).

The sequencing results did not turn out well. The sequencing locations aren't where they should be. After looking at these results with Andrey, we determined that the first modification to modify the NotI region appeared to have worked well. However, the 2nd modification to add the poly T near the Lac site did not appear to have worked well. We found that the reverse In-Fusion primer was incorrect since it excluded the poly Ts added during the 1st modification. The reverse sequencing primer (SAPTR) was also incorrect. Once I order these primers, and redo the in-fusion reaction, I may obtain the sequencing results I expect. Here's the sequencher file Andrey generated: "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\pcomb in-fusion model.SPF"

Some of my analysis can be found here. note that TL1, TL2, and MN are 3 different plasmids. The TL1 and TL2 plasmids should be plasmids after the "MN" (modify NotI) and the "TL" (add poly T near Lac promoter) modifications have been made. The MN plasmid should just be a plasmid after the "MN" modification only. alignment of TL1 at TL site (TL = add poly T near LacI) "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\alignment of TL1 TL site.txt" sequences at totally wrong location

alignment at TL2 at MN site is not good enough to make anything out because the sequences are too bad.

alignment of TL2 at TL site "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\alignment of TL2 TL site.txt" sequences at totally wrong location

alignment of TL2 at MN site (MN = modify NotI site) "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\alignment of TL2 MN site.txt" sequences at totally wrong location

alignment of MN at MN site "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\alignment of MN at MN site.txt" the SRNotI sequence does align with the poly T region as expected.

alignment of MN at TL site indicates that the poly T was not introduced just as expected. "L:\storage\CIM Research Folder\DR\2012\7-26-12\pcomb sequence analysis\alignment of MN at TL site.txt"

Now I need to order a new RPTF primer, RPTF2 primer, SAPTF, and SAPTR primer since Andrey identified the mistakes in my current primers. The planning document for these primers can be found here: "L:\storage\CIM Research Folder\DR\2012\6-13-12\inserting polyT site corrected.svg" and here "L:\storage\CIM Research Folder\DR\2012\7-29-12\pcomb\corrected primer check 7-29-12.SPF" The oligo order form can be found here "L:\storage\CIM Research Folder\DR\2012\7-29-12\pcomb\Oligo Order Kurt 7-29-12.xls" as well as here "S:\Research\Cancer_Eradication\Discovering tumor specific antigens\Phage Library\7-29-12 pcomb modifications"

8-15-12 I made a combined oligo order form to order the primers for making these phagemid pcomb modifications as well as for adding the poly T site to the cDNA library directional SMARTer In-Fusion library vector (see Designing primers to add poly T 8-5-12)

I combined the oligos from these two spreadsheets Oligo Order Kurt 7-29-12 ("L:\storage\CIM Research Folder\DR\2012\7-29-12\pcomb\Oligo Order Kurt 7-29-12.xls") Adding poly T primers (L:\storage\CIM Research Folder\DR\2012\8-15-12\adding poly T primers)

into this spreadsheet Oligo Order Kurt 8-15-12.xls ("L:\storage\CIM Research Folder\DR\2012\8-15-12\Oligo Order Form 8-15-12\Oligo Order Kurt 8-15-12.xls")

These oligos will be for modifying the phage pcomb plasmid RPTFC RPTFC2 SAPTFC SAPTRC RPTFCB RPTFC2B

These oligos will be for modifying the directional SMARTer In-Fusion library Lin1 Lin2 ATNL1 ATNL2