RNA

Part:BBa_K2889002:Design

Designed by: Rui Zhou   Group: iGEM18_Worldshaper-Wuhan   (2018-10-04)
Revision as of 10:55, 11 October 2018 by Ruizhou (Talk | contribs) (→‎Design Notes)

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pSB1C3-IL7-AS-S1


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 600
    Illegal SpeI site found at 813
    Illegal SpeI site found at 924
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 626
    Illegal SpeI site found at 813
    Illegal SpeI site found at 924
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 600
    Illegal SpeI site found at 813
    Illegal SpeI site found at 924
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 600
    Illegal SpeI site found at 813
    Illegal SpeI site found at 924
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We wanted to investigate which domain is essential for the function of IL7-AS. We cloned the truncated sequences of IL7-AS (IL7-AS-S1) into pSB1C3 for submitting to IGEM 2018 and inserted IL7-AS-S1 into pCDNA3.1 to study the function.

1.1 Amplification of IL7-AS-S1 fragments. First we amplified IL7-AS-S1 fragment (line 3) using primers. After that, we purified the PCR products by PCR Purification Kit and digested them with EcoRI and PstI (Fig 1).

Amplification_of_IL7-AS-S1.jpeg


1.2 Digested pSB1C3 vector. We digested the pSB1C3 vectors with EcoRI and PstI (Fig 2).

Digested_the_PSB1C3_vectors_with_EcoRI_and_PstI.jpeg


1.3 Ligation of purified IL7-AS-S2; IL7-AS-S1 and IL7-AS fragments to PSB1C3 vector. IL7-AS-S2; IL7-AS-S1 and IL7-AS fragments were ligated to PSB1C3 vector, respectively. Then we selected the positive clones by PCR and sequencing (Fig 3).

Verify_pSB1C3-IL7-AS-S1_by_sequencing.jpeg

Source

We cloned the truncated sequences of IL7-AS (IL7-AS-S1, 1099 bp) from human cells

References