BAB V KESIMPULAN DAN SARAN
5.2. Saran
Saran untuk penelitian selanjutnya perlu dilakukan sekuensing ulang terhadap plasmid rekombinan untuk mendapatkan sekuen susunan fragmen gen lipase dan dapat analisis tingkat homologinya dengan lipase golongan HSL yang telah dipublikasi. Sehingga, dapat dilakukan penelitan selanjutnya untuk mendapatkan susunan gen lengkap penyandi lipase golongan HSL dengan
menggunakan Thermal Asymmetric Interlaced PCR dan primer arbitrary
degenerate serta dilakukan kloning gen hingga ekspresi untuk karakterisasi enzim lipase golongan HSL dari tanah limbah kelapa sawit.
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LAMPIRAN Lampiran 1. Pembuatan larutan stok
Pembuatan larutan stok 0,1 M Tris-HCl pH 7.0 (Mr Tris = 121,14 gr/mol)
0,1 M =Mr Tris ×gram 100 ml1000 0,1 M =121,14 ×gram 100 ml1000 gram = 1,2114
Sebanyak 1,2114 gr Tris dilarutkan terlebih dahulu dalam 70 ml aquades lalu ditambahkan HCl 4N hingga pH 7.0 dan ditambahkan aquades kembali hingga 100 ml.
Pembuatan larutan stok 0,1 M EDTA pH 8.0 (Mr EDTA = 292,24 gr/mol)
0,1 M =Mr EDTA ×gram 100 ml1000 0,1 M =292,24 ×gram 100 ml1000 gram = 2,9224
Sebanyak 2,9224 gr Tris dilarutkan terlebih dahulu dalam 70 ml aquades lalu ditambahkan NaOH 4N hingga pH 8.0 dan ditambahkan aquades kembali hingga 100 ml.
Pembuatan larutan stok buffer fosfat pH 7.0
Dibuat larutan A yang mengandung 1 M NaH2PO4 (13,8 gr dalam
100 ml), dan larutan B yang mengandung 1 M Na2HPO4 (14,2 gr dalam 100
ml). Untuk mendapatkan bufer dengan pH 7.0, campurannya seperti tabel berikut.
A ( X ml ) B ( Y ml ) pH
39 61 7.0
Pembuatan larutan stok glukosa 0,1 M (Mr Glukosa = 180,16 gr/mol)
0,1 M =Mr Glukosa ×gram 100 ml1000 0,1 M =180,16 ×gram 100 ml1000 gram = 1,8016
Sebanyak 1,8016 gr glukosa dilarutkan dengan 100 ml aquades. Lampiran 2. Pembuatan larutan denaturan
10 mM Tris-HCl pH 7.0
V1 × M1 = V2 × M2
V1 × 0,1 M = 100 ml × 0,01 M
V1 = 10 ml (Tambahkan aquades hingga 100 ml)
1 mM EDTA
V1 × M1 = V2 × M2
V1 × 0,1 M = 100 ml × 0,001 M
V1 = 1 ml (Tambahkan aquades hingga 100 ml)
0,5% 2-Merkaptoetanol
Sebanyak 2,5 ml dari stok larutan 2% 2-merkaproetanol diencerkan dengan menambahkan aquades hingga 10 ml.
Lampiran 3. Pembuatan larutan buffer ekstraksi DNA
100 mM buffer fosfat pH 7.0
V1 × M1 = V2 × M2
V1 × 1 M = 100 ml × 0,1 M
V1 = 10 ml (Tambahkan aquades hingga 100 ml)
100 mM Tris-HCl pH 7.0 100 mM EDTA pH 8.0 1,5 M NaCl (Mr = 58,44 gr/mol) 1,5 M =Mr NaCl ×gram 100 ml1000 1,5 M =58,44 ×gram 100 ml1000 gram = 8,766
Sebanyak 8,766 gr NaCl dilarutkan dengan 100 ml aquades.
1% CTAB
Sebanyak 1 gr CTAB dilarutkan dalam 100 ml aquades.
2% SDS
Lampiran 4. Desain primer CLF dan CLR untuk amplifikasi fragmen gen lipase
Lampiran 5. Pembuatan larutan untuk isolasi plasmid
Larutan I
o 50 mM glukosa
V1 × M1 = V2 × M2
V1 × 0,1 M = 100 ml × 0,05 M
V1 = 50 ml (Tambahkan aquades hingga 100 ml)
o 25 mM Tris-HCl pH 8.0
V1 × M1 = V2 × M2
V1 × 0,1 M = 100 ml × 0,025 M
V1 = 25 ml (Tambahkan aquades hingga 100 ml)
o 10 mM EDTA pH 8.0
V1 × M1 = V2 × M2
V1 × 0,1 M = 100 ml × 0,01 M
V1 = 10 ml (Tambahkan aquades hingga 100 ml)
Larutan II
o 0,2 M NaOH
V1 × M1 = V2 × M2
V1 × 4 M = 100 ml × 0,2 M
V1 = 5 ml (Tambahkan aquades hingga 100 ml)
o 1% SDS
Larutan III
o 5 M Na-asetat (Mr = 82,0343 gr/mol)
5 M =82,0343 ×gram 20 ml1000 gram = 8,2034