Using Bioedit to Refine DNA Sequences

Getting the Desired Portion of DNA

1. Open the Forward and Reverse ab1 files for your sequence with Bioedit.
2. Find the window with the Forward sequences chromatogram waves. If you have a clean
sequence, you should see clear, unambiguous peaks representing A, T, C and G. Use the
scroll bar on the left to adjust the height of the waves if you have trouble seeing them.
3. Scroll to the right until the chromatogram waves come to an end (around 410 base pairs).
The sequence generated beyond this point is junk DNA. Make a note of where your
sequences end point is.
4. Find the window with the Forward sequence in fasta format (just letters, no
chromatogram waves).
a. Change the mode from Select/Slide to Edit in the drop-down menu in the
upper left-hand corner.
b. Highlight everything beyond the end point of your desired sequence and delete it.
**You can manually click and drag to select everything, or:
i. click on the desired end point
ii. go to select to end under the Edit feature in the top tool bar
5. Repeat steps 2-4 with the Reverse sequence.
Putting Forward and Reverse Sequences Together
1. Select the Reverse fasta form file name from the left hand side (ex. BR_3.g1) and press
Shift+Ctrl+R to generate a reverse complement strand. Now the forward and reverse
sequences are running in the same direction and have (mostly) the same nucleotides.
2. Double click on the file name to the left of the sequence to open a new editing window.
3. Highlight and copy the entire sequence (Ctrl+C)
4. Go to the Forward sequence fasta window. Select new sequence under the Sequence
feature in the top tool bar.
a. Paste your Reverse sequence in the new window (Ctrl+V)
b. Rename this new sequence R in the Name field.
c. Select DNA for Sequence Type to get the appropriate nucleotide colors.
d. Select Apply and Close. Now both sequences should show up in your Forward
window.

Alignment Editing
1. Under the Accessory Application field in the top tool bar, select ClustalW multiple
alignment. This program will match up your two strands.
a. Click on Run ClustalW and OK when program windows pop up.
2. Mouse over the colorful icons representing the different view modes. Select shade
identities and similarities. This makes it easier to see which parts of your Forward and
Reverse sequences match up.
3. Start editing inconsistencies from the end of the sequence. Refer back to the
chromatograms to see which nucleotide wave is more prominent or reliable for each
ambiguity. (This can be tricky if your sample is contaminated.)
a. In your chromatogram window for the Reverse sequence, select Reverse
Complement under the View feature in the top tool bar. This allows you to see
waves for the reverse complement. (The first several hundred will be junk DNA
that used to be at the end of the sequence.)
b. To locate the regions in question in the chromatogram window, copy/paste a small
portion of the aligned sequence into the search query. (Ctrl+F)
c. Make sure you keep track of which sequence you are looking at when checking
base pairs!
4. Delete first 10 base pairs from the beginning of your sequence (where the primers
annealed).
5. When you are finished, double click the file name for either the top or bottom row (they
are identical) to open up a separate editing window.
a. Copy and paste this new sequence into Microsoft Word and save it to use for
BLAST.

Menggunakan Bioedit untuk Pertajam DNA Urutan

Mendapatkan Bagian Diinginkan DNA
1. Buka forward dan Reverse file ab1 untuk urutan Anda dengan Bioedit.
2. Cari jendela dengan gelombang kromatogram urutan Forward. Jika Anda memiliki urutan
bersih, Anda harus melihat jelas, puncak ambigu mewakili A, T, C dan G. Gunakan
scroll bar di sebelah kiri untuk menyesuaikan ketinggian gelombang jika Anda
memiliki kesulitan melihat mereka.
3. Geser ke kanan sampai gelombang kromatogram berakhir (sekitar 410 pasangan basa). Urutan
yang dihasilkan melebihi titik ini masih DNA sampah. Buat catatan dari mana titik
akhir urutan Anda adalah.
4. Cari jendela dengan urutan Teruskan dalam format fasta (hanya huruf, tidak ada gelombang
kromatogram).
Sebuah. Mengubah mode dari "Pilih / Slide" untuk "Edit" pada menu drop-down di sudut kiri
atas.
b. Sorot segala sesuatu di luar titik akhir urutan yang Anda inginkan dan menghapusnya.
** Anda dapat secara manual klik dan drag untuk memilih semuanya, atau:
saya. klik pada titik akhir yang diinginkan
ii. pergi ke "pilih untuk mengakhiri" di bawah fitur "Edit" di tool bar atas
5. Ulangi langkah 2-4 dengan urutan Lookup.
Letakkan Urutan Forward dan Reverse Bersama
1. Pilih Reverse bentuk fasta nama file dari sisi kiri tangan (ex. BR_3.g1) dan tekan Shift + Ctrl
+ R untuk menghasilkan pelengkap strand terbalik. Sekarang maju dan mundur urutan
berjalan di arah yang sama dan memiliki (kebanyakan) nukleotida yang sama.
2. Klik dua kali pada nama file di sebelah kiri urutan untuk membuka jendela editing baru.
3. Sorot dan copy seluruh urutan (Ctrl + C)
4. Buka jendela urut fasta Teruskan. Pilih "urutan baru" di bawah fitur "Urutan" di tool bar atas.
Sebuah. Tempel urutan balik Anda di jendela baru (Ctrl + V)
b. Ubah nama urutan baru ini "R" di bidang "Nama".
c. Pilih "DNA" untuk "Urutan Type" untuk mendapatkan warna nukleotida yang sesuai.
d. Pilih "Apply dan Tutup." Sekarang kedua urutan harus muncul di jendela Teruskan Anda.