Team:Heidelberg/Templates/Del week16 overview

From 2013.igem.org

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Latest revision as of 18:38, 4 October 2013

Contents

Strategy

Vector map of including Primers FS_01 to FS_16 used for assembly of the desired genes from D. acidovorans and the partsregistry backbone pSB4K5 .

Most PCRs have worked!! Yeah- finally!!! Thus, all the fragments needed for Gibson assembly are now available, however yields are still suboptimal for some of the fragments amplified. Furthermore, we designed screening primers for our final construct pFSN. These primers will be used to analyze our transformed bacteria for presence of the complete DelRest construct pFSN by colony-PCR.



Primer pairs, corresponding sequences and usage

Identifier Order date Note Sequence
FS_47_screening_BB_AF_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTTGGCCGATTCATTAATGC
FS_48_screening_BB_AF_rv2013-08-02 Primer for screening/sequencing of pFSN construct TAACGGTATCGGTATCGCTTTG
FS_49_screening_AFI_AFII_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTTTCTCTGGAAGATGGATAC
FS_50_screening_AFI_AFII_rv2013-08-02 Primer for screening/sequencing of pFSN construct GTTGACGAAAAAGCCGACCAC
FS_51_screening_AF_FG(21-26)_fw2013-08-02 Primer for screening/sequencing of pFSN construct TGGATATCGACTGGACTGCCTG
FS_52_screening_AF_FG(21-26)_rv2013-08-02 Primer for screening/sequencing of pFSN construct TGCACCACATCGACGAAACGG
FS_53_screening_FG(21-26)_G_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTACGGCCTATCACATCAGCG
FS_54_screening_FG(21-26)_G_rv2013-08-02 Primer for screening/sequencing of pFSN construct GAACCTGGGTGTTCACGAAAAAGCC
FS_55_screening_G_OP_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTATCTCTACATGCATCGCTAC
FS_56_screening_G_OP_fw2013-08-02 Primer for screening/sequencing of pFSN construct AGGACATTTTCCGCACCCCG
FS_57_screening_G_OP_rv2013-08-02 Primer for screening/sequencing of pFSN construct GCTGGCGTTTTCCATAAG
FS_58_screening_OP_L_fw2013-08-02 Primer for screening/sequencing of pFSN construct GAACAACTTCCAGCACAGCCTGTTC
FS_59_screening_OP_L_rv2013-08-02 Primer for screening/sequencing of pFSN construct CGTTGAAGATTTCGTTGACG
FS_60_screening_L_BB_fw2013-08-02 Primer for screening/sequencing of pFSN construct CATCTTCAAGGTGTTCTATGAAC
FS_61_screening_L_BB(with_mRFP)_rv2013-08-02 Primer for screening/sequencing of pFSN construct CAGTTTAACTTTGTAGATGAAC
NK_01_FS_62_screening_L_BB(without_mRFP)_rv2013-08-02 Primer for screening/sequencing of pFSN constructGTTCACCGACAAACAACAGATAAAACG

Amplification and purification of Del Genes from SPH-1

Goals

As we were able to establish amplification protocols for most of our desired amplicons by the end of last week, we will repeat these PCRs to obtain the required amounts and concentrations. Furthermore we will optimize the PCR conditions of DelO-P by using higher annealing temperatures, hopefully inhibiting secondary structe formation of the primers. Afterwards we will assemble our construct, transform it into E.Coli DH10B and screen for (hopefully many) positive clones using the recently ordered screening primers.

Results

PCRs from D.acidovorans SPH-1
Gene(s) Fragment Primer combination Successful?
DelA
DelB
DelC
DelD
DelE
DelF
DelG
DelA-F Primer FS_02 and FS_05
Heidelberg Yes check.svg.png
DelA-G Primer FS_02 and FS_11
Heidelberg Red x.svg.png
Primer FS_02 and FS_11_short
Heidelberg Red x.svg.png
DelF-G Primer FS_21 and FS_07
Heidelberg Tilde.png
Primer FS_21 and FS_26
Heidelberg Yes check.svg.png
DelG Primer SR_01 and FS_23
Heidelberg Yes check.svg.png
DelO
DelP

DelL
DelO-P Primer FS_22 and FS_13_short
Heidelberg Yes check.svg.png
Primer FS_22 and FS_13
Heidelberg Yes check.svg.png



Test restriction digests of PCR amplified fragments
Fragment Primer Digestion enzyme Expected bands
DelO-P Primer FS_22 and FS_13 EcoRI-HF 1883, 960