Team:USyd-Australia/Notebook/Primers

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Primers

All primers were ordered from New England Biolabs.

Primers were reconstituted in TE buffer to a stock concentration of 500μM. This is to minimise the number of freeze-thaw cycles that the primers undergo when being used. Dilutions were made to produce working primer stocks of 50μM or 10μM as required.

To make 500μM stocks, Volume to add=(2x number of nMoles) μL

Below is a table of sequences of all primers used in the USyd-Australia 2014 iGEM project, accompanied by a brief description of its application

NameSequence (5'-3')Theoretical TmPurpose
iGEM10AGCTTTCGCTAAGGATGATTTCTGGA58.2CScreening and sequencing
iGEM16GAGTGCCACCTGACGTCTAAGAAACC60.9CFor BioBrick sequencing, amplification by PCR, or junction PCR. Alternative to iGEM VF2 primer
iGEM25CCCTGATTCTGTGGATAACCGTATTACCG60.0CFor BioBrick sequencing, amplification by PCR, or junction PCR. Alternative to iGEM VR primer
iGEM1401GGGAAGCTTTCTTAGACGTCAGGTGGCACTTTTCGG66.3C pBBR replicon
iGEM1402TTTGGATCCTATGCTGCTGGCTACCCTGTGGAAC66.5CpBBR replicon
iGEM1403GTTCCACAGGGTAGCCAGCAGCATAGGATCCAAAGA
GTGCCACCTGACGTCTAAGAAACC		71.2Cprimers for pSB prefix/suffix
iGEM1404CCGAAAAGTGCCACCTGACGTCTAAGAAAGCTTCCC
CCTGATTCTGTGGATAACCGTATTACCG		70.3primers for pSB prefix/suffix
iGEM1405GAGGTCGGACAGTATATTGAAACGTCAGGAGTCTAG
ATACCGAGGGACAGACTACCAACTCACA		 primers for gene cassette circularisation (attC-aeBlue-aacC1)
iGEM1406 GTATCTAGACTCCTGACGTTTCAATATACTGTCCG
ACCTCTTATTAGGTGGCGGTACTTGGGTCG		 primers for gene cassette circularisation (attC-aeBlue-aacC1)
iGEM1407 CTATATCTATGATCTCGCAGTCTCC 53.8C PCR primers over the junction between aacC1 (G block) and pSB1C3 vector, for validation of successful G block insertion by PCR screening
iGEM1408GCCTTTTGCTCACATGTTCTTTC55.2CPCR primers over the junction between aacC1 (G block) and pSB1C3 vector, for validation of successful G block insertion by PCR screening
iGEM1409 GTTTTTTTGGATGGAGTGAAACGATGGCGATTG 61.7C Amplification of iGEM-IntI1 gBlock forward primer
iGEM1410 TGACACCTTGCCCTTTTTTGCCG 60.9C Amplification of iGEM-IntI1 gBlock reverse primer
iGEM1411 GTTTTTTTGGATGGAGTGAAACGATGGCGATTG 61.7C Gblock primers for IntI1 in pSAM-R
iGEM1412 TGACACCTTGCCCTTTTTTG 53.9C Gblock primers for IntI1 in pSAM-R
iGEM1413 GACATTGCCGTCACTGCGTC 59.1C Junction primers for IntI1 in pSAM-R
iGEM1414 TGGTCCAGAACCTTGACCGAAC 59.2C Junction primers for IntI1 in pSAM-R
iGEM1415 AACCGAGGATGCGAACCACTTC 59.7C Junction primers for IntI1 in pSAM-R
iGEM1416 CCTTCAAACGTGCCGTAGAACAAGC 60.4C Junction primers for IntI1 in pSAM-R
iGEM1417 AAATACTAGTAGCGGCCGCTGCAGTCCGGCAAAA
AAG 		67.6C Linearisation of pSB1C3, containing full BioBrick prefix and suffix
iGEM1418 AAACTCTAGAAGCGGCCGCGAATTCCAGAAATCA
TCCTTAGC 		66.9C Linearisation of pSB1C3, containing full BioBrick prefix and suffix
iGEM1419 TTTGAATTCGCGGCCGCTTCTAGAGTACCGAGGG
ACAGAC 		68.9C Amplification of aeBlue gBlock. Forward primer.
iGEM1420 GGGCTGCAGCGGCCGCTACTAGTATTATTAGGTGG 67.4C Amplification of aeBlue gBlock. Reverse primer.
iGEM1421 GGGAATTCAAATCTAGAGACACCATCG 57.1C Amplification of LacI-Plac-AttI gBlock. Forward primer
iGEM1422 CCTGCAGTTTACTAGTTTTGCCTAACTTTGTTTTAG 59.4C Amplification of LacI-Plac-AttI gBlock. Reverse primer
iGEM1423 CAGGCTTTACACTTTATGCTTCCG 56.3C lacI-Plac-attI RHJ-F right hand junction forward primer (use with iGEM25)
iGEM1424 AATGTAATTCAGCTCCGCCATC 55.5C lacI-Plac-attI LHJ-R left hand junction reverse primer (use with iGEM16)
iGEM1425 AAATCTAGATGGCGCGGCTTAACTCAGGTGTTAG
GCTTAGGAGGCTCAAGTATGGGCAT 		70.7C For making aacC1 gene cassettes from Tn1696. Use with iGEM1426.
iGEM1426 AAAACTAGTGGCGTCGGCTTGGACGAATTGTTAG
GCTTAGGTGGCGGTACTTGGGTC 		71.4C For making aacC1 gene cassettes from Tn1696. Use with iGEM1425.
iGEM1427 TTTTCTAGAGTTTGATGTTATGGAGCAGCAACG 60.2C For making aacC1 gene cassettes from Tn1696. Use with iGEM1428. Alternative to iGEM1424.
iGEM1428 TTTACTAGTGCAAAAAGGCAGCAATTATGAGCC 60.9C For making aacC1 gene cassettes from Tn1696. Use with iGEM1427. Alternative to iGEM1426.
NVC71 CTAAGAATCCATAGTCCAACTCC 52.4C aadB gene, rvs primer for junction screen
NVC92b CACGCAAGACCTCAACCTTTTCC 58.6C aadB gene, rvs primer for junction screen
NVC158 GATACCTTGTGCGGCTATGTCTG 57.6C pUS41/44, left of cloning site
NVC159 GCCGCCTTGGGCCGGGTGATGTC 69.2C pUS41/44, right of cloning site

With thanks to:

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