User:Jessica Karen Wong/Primers

Scarring Primers
These will be used to PCR out T9002 in order to scar out the BB sites from a backbone plasmid.
 * T9002_F -- CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATC
 * T9002_R -- TCAGCCAT ATGCAT TATAAACGCAGAAAGGCCCAC

Bold is the tail, italics is the restriction site.

Bold is the tail, italics is the restriction site.
 * New T9002 Reverse that matches only GFP TCAGCCAT ATGCAT TTATTATTTGTATAGTTCATCCATGCC
 * Melting Temp 55.0
 * Original T9002_F CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATC has melting temp 53.9


 * New Longer I2055
 * Fwd- CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATCCCTATC melting temp 62
 * Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCCCACCCG melting temp 63.6


 * New Shorter I2055
 * Fwd- CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTG melt 45.4
 * Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCC melt 45.5

Keep same tails?


 * Original I2055
 * Fwd- CTTAGTAG CAATTG tccctatcagtgatagagattgacatc melt 53.9
 * Rev- TCAGCGAT ATGCAT TATAAACGCAGAAAGGCCCAC melt 53.6 (is actually same primer as E0240R)
 * I2056:
 * F: Same as I2055
 * R: same as I2055


 * I2057:
 * F: CTTAGTAG CAATTG TCACACAGGAAAGTACTAGATGGC (52.1)
 * R: same as I2055

Also designed primers to PCR R0040 in if none of the colonies were successfully ligated

* Only ordered a forward primer with Tail including Mfe1 and R0040 * format: 8-mer.MfeI.promoter+mix.part of gfp (Tm) * CTTAGTAG.CAATTG.tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactag.ATGCGTAAAGGAGAAGAACTTTTC (52.4)

Ordered new I2056 primers to PCR in Promoter
 * I2056_R0040_F 8mer.Mfe1.promoter+mix.part of RFP
 * CTTAGTAG CAATTG tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactag ATGGCTTCCTCCGAAGACG (54.0)

BioBrick Primers
* E0240: o F (incl E0240, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGCG (53.1) o R (incl 3K3/1AK3, EcoRI): TCAGCGAT.GAATTC.CAGAAATCATCCTTAGCGAAAGC (54.2)

* I2057: o F (incl I2057, PstI): CTTAGTAG.ACTAGT.TCACACAGGAAAGTACTAGATGGC (52.1) o R: same as E0240

* I2055: o F (incl GFP, SpeI): CTTAGTAG.ACTAGT.ATGCGTAAAGGAGAAGAACTTTTC (52.4) o R (incl R0040, EcoRI): TCAGCGAT.GAATTC.GTGCTCAGTATCTCTATCACTGATAGG (52.0)

* I2056: o F (incl RFP, SpeI): CTTAGTAG.ACTAGT.ATGGCTTCCTCCGAAGACG (54.0) o R: Same as I2055

* For T9002: o F (incl RBS, GFP,EcoRI): TCAGCGAT.CTGCAG.TCACACAGGAAAGTACTAGATGCG (53.1) o R (incl lux pR, PstI) : CTTAGTAG.GAATTC.TTTATTCGACTATAACAAACCATTTTC (51.9) Format: 8-mer.restriction site.primer

New Primers
T9002 pieces


 * F2620-F    8mer mfe1 TCCCTATCAGTGATAGAGATTGACATC 53.9
 * CTTAGTAG CAATTG TCCCTATCAGTGATAGAGATTGACATC


 * F2620-R    8mer xba1 not1 eco1 TTTATTCGACTATAACAAACCATTTTC 51.9
 * TCAGCGAT CTCTAGAAGCGGCCGCGAATTC TTTATTCGACTATAACAAACCATTTTC


 * E0240-F    8mer spe1 NOT1 pst1 TCACACAGGAAAGTACTAGATGCG 53.1
 * CTTAGTAG tactagtagcggccgctgcag TCACACAGGAAAGTACTAGATGCG


 * E0240-R    8mer nsi1 GTGGGCCTTTCTGCGTTTATA 53.6
 * TCAGCGAT ATGCAT GTGGGCCTTTCTGCGTTTATA

I2055/I2056 to account for RBS spacing


 * I2055-F 8mer Xba1 ATGCGTAAAGGAGAAGAACTTTTC 52.4
 * CTTAGTAG TCTAGA TGCGTAAAGGAGAAGAACTTTTC


 * I2055-R 8mer xba1 NOT1 ecor1 GTGCTCAGTATCTCTATCACTGATAGG 52.0
 * TCAGCGAT CTCTAGAAGCGGCCGCGAATTC GTGCTCAGTATCTCTATCACTGATAGG


 * I2056-F 8mer Xba1 ATGGCTTCCTCCGAAGACG 54.0
 * CTTAGTAG TCTAGA TGGCTTCCTCCGAAGACG


 * I2056-R same as I2055-R

Nishant's Primers
1) TCAGCGAT GCGGCCGC GAATTC CAGAAATCATCCTTAGCGAAAGC, BB_P_Rev_Eco

2) CTTAGTAG GCGGCCGC ACTAGT TCACACAGGAAAGTACTAGATGCG, BB_E0240_Fwd_Spe

3) CTTAGTAG GCGGCCGC TCTAGA TCACACAGGAAAGTACTAGATGCG, BB_E0240_Fwd_Xba

4) TCAGCGAT GCGGCCGC ACTAGT GTGCTCAGTATCTCTATCACTGATAGG, BB_R_Rev_Spe

5) TCAGCGAT GCGGCCGC GAATTC GTGCTCAGTATCTCTATCACTGATAGG, BB_R_Rev_Eco

6) CTTAGTAG GCGGCCGC TCTAGA ATGCGTAAAGGAGAAGAACTTTTC, BB_I2055_Fwd_Xba

7) TCAGCGAT GCGGCCGC GAATTC TTTATTCGACTATAACAAACCATTTTC, BB_T9002_Rev_Eco

8) CTTAGTAG GCGGCCGC GAATTC TCACACAGGAAAGTACTAGATGCG, BB_T9002_Fwd_Pst

9) CTTAGTAG GCGGCCGC TCTAGA ATGGCTTCCTCCGAAGACG, BB_I2056_Fwd_Xba

10) CTTAGTAG GCGGCCGC ACTAGT TCACACAGGAAAGTACTAGATGGC, BB_I2057_Fwd_Spe

11) CTTAGTAG GCGGCCGC TCTAGA TCACACAGGAAAGTACTAGATGGC, BB_I2057_Fwd_Xba

REVISION 7-23


 * CTTAGTAG GCGGCCGC ACTAGT G TCACACAGGAAAGTACTAGATGCG, BBbp_E0240_Fwd_Spe
 * CTTAGTAG GCGGCCGC TCTAGA G TCACACAGGAAAGTACTAGATGCG, BBbp_E0240_Fwd_Xba
 * CTTAGTAG GCGGCCGC ACTAGT G TCACACAGGAAAGTACTAGATGGC, BBbp_I2057_Fwd_Spe
 * CTTAGTAG GCGGCCGC TCTAGA G TCACACAGGAAAGTACTAGATGGC, BBbp_I2057_Fwd_Xba
 * TCAGCGAT GCGGCCGC ACTAGT A GTGCTCAGTATCTCTATCACTGATAGG, BBbp_R_Rev_Spe
 * CTTAGTAG GCGGCCGC TCTAG ATGCGTAAAGGAGAAGAACTTTTC, BBbp_I2055_Fwd_Xba
 * CTTAGTAG GCGGCCGC TCTAG ATGGCTTCCTCCGAAGACG, BBbp_I2056_Fwd_Xba