2022 pEC-ExcellScript
Cloning of 2022 pEC-ExcellScript
[edit]Purpose: Cloning of2022 pEC-ExcellScript==
Date: 2014-1----
Notebook:0
Page:
Clone ID:2022
Experimenters: shanyi
Results
[edit]CLONING
[edit]PCR
1. first PCR 7 fragments, do not amply more than 24 cycles, usually 20 cycles should be enough
ssIII-Nde1-F1
ctagcCATATGACCCTAAATATAGAAGATGAGCATCGGCTAC
ssIII-L52P-R1
(CTCCTCTGATCATACCT CcG AAAGCAACC TCTACCCCCGTGTCC)
GGACACGGGGGTAGAGGTTGCTTTCgGAGGTATGATCAGAGGAG
ssIII-Y64S-F2
G AAAGCAACC TCTACCCCCGTGTCC ATAAAACAA Tct CCg ATGTCACAAGAAG
ssIII-K152M-R2
(GTGCTTGATTTA AtG GATGCCTTT TTC)
GAAAAAGGCATCCaTTAAATCAAGCAC
ssIII-K152M-F3
GTGCTTGATTTA AtG GATGCCTTT TTC
ssIII-H204R-R3
(GAGGCACTG CgC AGA GACCTAGCAGACTTC CGGATCCAGCACCCA GAC)
GTCTGGGTGCTGGATCCGGAAGTCTGCTAGGTCTCTGcGCAGTGCCTC
ssIII-V223H-F4
CAGACTTCCGGATCCAGCACCCAGACTTGATCCTGCTACAGTACcatGATGACTTACTGCTG
ssIII-M289L-R4
(GAGGCCAGAAAA GAGACTGTG cTG GGG CAGCCTACTCCGAAG ACCCCTCGACAACTAAG)
CTTAGTTGTCGAGGGGTCTTCGGAGTAGGCTGCCCCAgCACAGTCTCTTTTCTGGCCTC
ssIII-T306K-F309N-F5
CTCCGAAG ACCCCTCGACAACTA AGGGAGTTCCTAGGG Aaa GCAGGC aaC TGT CGCCTCTGGATCCCT G
ssIII-D524G-R5
(CACC TGGTACACG GgT GGA AGCAGTCTCTTAC)
GTAAGAGACTGCTTCCAcCCGTGTACCAGGTG
ssIII-D524G-F6
CACG GgT GGA AGCAGTCTCTTAC
ssIII-E562Q-R6
(CTCAGCGG GCT cag CTGATAGCA CTCACCCAGGCCCTA AAGATGGCAGAAG)
CTTCTGCCATCTTTAGGGCCTGGGTGAGTGCTATCAGctgAGCCCGCTGAG
ssIII-D583N-F7
CAGGCCCTA AAGATGGCAGAAGGT AAGAAGCTAAATGTT TATACT aAT AGCCGT TATGCTTTTG
ssIII-Xho1-R7
(ctaccctcctcatagaaaattcatca ccg aattcccgcttaattaat ctcgag taacgc)
gcgttactcgagattaattaagcgggaattcggtgatgaattttctatgaggagggtag
2. Purify these fragments (mix gel slices, add QG, dissolve, purify using one spin column).
Use purify DNA mix as template, do PCR:
ssIII-Nde1-F1
ctagcCATATGACCCTAAATATAGAAGATGAGCATCGGCTAC
ssIII-Xho1-R7
(ctaccctcctcatagaaaattcatca ccg aattcccgcttaattaat ctcgag taacgc)
gcgttactcgagattaattaagcgggaattcggtgatgaattttctatgaggagggtag
digest full length PCR product with Nde1 + Xho1, then ligate to Nde1 + Xho1 digested vector
Colony Screening
[edit]Date: 2013-10-23
Make miniprep, digest with Nde1 + Xho1+BamHI