* 1. Which strain should be used to express this protein?

atg ctg aga ggc tca aca gga att tac tgt atg cag gaa gat gcc ggg aga att act agg acc aga cat gtg gat cgt gta acc att ccc aag aaa aga ata tta aac gaa gga gct cac gag tat gat gga ttg tca ggc ggt tca act tca gct cag gat gta gtc aaa aac ctt ttg gca atg aaa gaa aaa gaa ttt tgt aca aga cat cct att ctt gca tat aca aac ata gta gag aca caa gag cat gta att gat cta ata aac cta gca acc ata gtt ctg ggg cgt ctt ggt cct ttt caa gag gca aaa aat aat gat ggt aat atc gaa gtt gtg ggt gat act agt gat aaa caa aaa tta gat cca ggt ttg aat ttc agc gtt gtg tat cta ttt caa ttc ttt tta ttt ccc aaa cag tcc gtg act ggt cca agg gta ttt aca aag cta aca act gta gga gga aag gtg aac act aat ggt atc aac act ggc tgt aat gga tta atc aca gga gat tta aag ata taa

* 2. This protein has been just eluted from a Ni column using Ni Column Elution Buffer. We want to remove endotoxin from the partial purified protein. Please design an effective purification method without using an expensive resin and equipments:

1). Loading Buffer for loading protein onto a column:
2). Washing Buffer:
3). Elution Buffer:
4). Column:



Ni Column Elution Buffer: 20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole, 0.1 mM EDTA, 1 mM beta-ME.


Protein:

M A H H H H H H R V T P Q R R T D M W N K V Q R S S E T A R R Q S S F L A L L A D V V R G A P M C S G H T V A I G N K G S M H G V E I M R A M K D N G E V I E D P D V S Q A R D N L T E R A D L K D L R L V T E G R T A E R T F I E D L K R S K C D D R A A S E A R Q P I K V S Q N P V R Q R L G M Y L A G L Q E A D M W S Q G H L L A I S V A L G R H L L K R A L Y M S L E V V A N R H L V T F W W N D R S L R G T D A V W L K L D A S Q M N L F I D L N I P R A S Y E G R Q D F I R G S Y F A K G F L R R A I V H S G L A R E N R N N A H S A K P D L P K N G L S A L R S R V V Q P N M A I V G V L A Y I I N D A A P K G G K D T M L R P C N L S E T G L D I I W R E E D R Y I N T Y L G F G N S I K T K L V T V W A L V K A A S C R V T

* 3. You loaded a partial purified protein (DNA ligase, Peak 1) onto a 5ml HiTrap Q FF column. The chromatogram is shown below. Please design a method to remove contaminating E coli DNA after eluting the protein from a Ni column using Ni Column Elution Buffer (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 300 mM imidazole, 1 mM beta-ME).

Anion Exchange Chromatography

Anion Exchange Chromatography

* 4. Please design an enzyme mix for preparing PCR template damaged DNA

* 5. You loaded 50 ml of BL21 lysate containing 50 mg of 6XHis tagged protein in Bind Buffer to 10 ml of Ni-NTA resin. You washed the column with 500 ml of Wash Buffer, and eluted protein with 30 ml of Elute Buffer. How much protein should you get?

Bind Buffer (use for lysis and binding): 50 mM Tris-HCl, pH 8.0; 300 mM NaCl; 10 mM imidazole
Wash Buffer: 50 mM Tris-HCl pH 8.0; 300 mM NaCl; 1 mM EDTA; 20 mM imidazole
Elute Buffer: 50 mM Tris-HCl, pH 8.0; 300 mM NaCl; 250 mM imidazole

* 6. Check Ni-NTA compatible reagents

* 7. Based on the following public information, please design primers to clone ATP-dependent exonuclease, (AKA Plasmid Safe DNase) from Micrococcus luteu.

http://www.sciencedirect.com/science/article/pii/0005278781901180
http://www.ncbi.nlm.nih.gov/pubmed/4355370
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC427131/
https://www.jstage.jst.go.jp/article/biochemistry1922/92/4/92_4_1205/_article
http://www.ncbi.nlm.nih.gov/nuccore/239916571?report=genbank

* 8. Design oligos to synthsize this DNA fragment by PCR (gene synthesis)
AGATCtGGAATTGTGAGCGCTCACAATTCCCTTTCCCCACAACGGAACAACTCTCATTGCATGCTTTCCCCACAACGGAACAACTCTCATTGCATGTGGGAATTGTGAGCGCTCACAATTCCCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTGCACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTAATGGCTGCATTCTGCGGTAAGCACGAACTCAGCCAGAACGACAAACAAAAGGCTATCAACTATCTGATGCAATTTGCACACAAGGTATCGGGGAAATACCGTGGTGTGGCAAAGCTTTATTAAGAGGTAATACACC
ATGAATCACAAAGTGctcgag

* 9. Design a buffer for purifying this protein:

MADAAQTQLTSFCTMHGEQNRLIERQETECEFGGYMQVLPDVGVAIQRNAEQNARTHVKDLKCDEGCVEVGCRFTTKAVGHKIYSPAVCDGYEVENESAVILMYASDGARLPYPSERYKCPRSTLITFCHVTAASEGLQSRGGTLETFVANDGEDAMSNGSGLPSDDAPMWNNQVPRRPITIGVFQSAESSTTFWCSQVDKHDIQCDPCQIRQVLLVTLIFGLNQSQVRMTDALFMVYHMNMAGCIWASSCEGDLVQSYFADKRGGIRQTRGHTVANVINAPREEERFRRAMLCSYYVASNTYNGMERVYELTWSPQRPAKELIEEREMHMGVQYFDNANEHEVLVISRMVGDGMLGLNIPTPSHIHRAVALKDRPSSAVGRCYLGVVAAIVVHVACHYAKNVLAHYDVVNCGSDLMQHREEGPSEGQLNQALFFRTYDGCIGPMGYHQYQLFVISLPLTVGDDPASVCAVNARGMGGLTGLHGTWGVYDDSLGCPPLSSLSANEATCALGYGSYLGVDISMVNRIWPRVNQRPKTSHQGLYTEELFCGVKIRFLQRYYILAVTSGLACSKPRKPVMQDDMQETQNLSFPCIHLLRCQLLVRKARVRHCWAEFEYQVAVYKICFNDQS

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