Handout 10/19/2015
Picking white colonies:
Every team member of the groups that performed a single cloning recations
(1,2,3; 5,6,7; 8,9,10).
Use sterile yellow tips to pick and deposit three separate white colonies in the medium (LB, 50mg?ml
kanamycin) provided.
Label tubes with your initials, group number, and tube number.
e.g.: CA, 4, 3.
Sequencher finalizing edits COI PHYSID snails.
In sequencher
1 Import the directseq_1 results and directseq_2 results into one project file (you may have already done
this)
2 Edit “your” sequences (for your snail, focus on COI sequences). If your sequences are not edit-able, select
Sequences for another sample.
Direct seq_1 samples:
Group
1
2
3
5
6
7
8
9
10
SAMPLE TARGET/PRIMER
16S
1
F(01)
2
F(04)
3
5
F(07)
4
F(09)
5
R(11)
4
R(13)
1
R(15)
2
R(19)
COI
R (02)
R(03)
F(05),R(06)
R(08)
R(10)
F(12)
F(14)
F(16)
F(18)
Directseq_2 samples are labled for snail sample and gene target
3 Remove start and trailing N residues. (CTRL-S) Save your work
4 Return to project window. Click on assembly parameters, check dirty data, minimum match percentage
85, minimum overlap 60. OK
5 Select all your sequences (derived from the same gene from your snail sample)
e.g. 02-1.2_CO1R; 16_G8_CO1F; 03-403S1C)IF; 04-404S1COIR.
6 click assemble automatically.
7 if no contig resulted, go back to assembly parameters and relax criteria.
If contig resulted, open contig and check your editing, can you improve the consensus sequence?
8 BLAST to check whether you have a snail sequence.
9 do a BLASTN (standard settings) and a BLASTX, make sure to select genetic code 5.
10 e-mail your consensus sequence to coenadem@unm.edu
INSERT CHECK BY RESTRICTION DIGEST
SET UP restriction digestion for insert check:
Label your tubes, group number, and 1 - 4 (e.g 1-1; 1-2; 1-3; 1-4)
Combine in a master mix:
4 microliter of EcoRI (storage buffers of restriction enzymes can inhibit reaction if >10% of final reaction volume)
6 microliter of 10x buffer
42 microliter of milliQ water, mix, aliquot 13 microliter to 4 tubes each.
Add 2 microliter of plasmid sample
Incubate 30 minutes, 37C.
Run 1% agarose gel
Inspect for presence absence of plasmids, inserts.
Home work (also if you did not run the gel)
1)
2)
3)
describe the basic functioning of the DEAE (Qiagen) method for purification of DNA.
Describe what banding pattern on the gel shows that the restriction digestion confirms the presence of a
plasmid with an insert.
Can you predict how many gel bands will result from the insert after restriction digest with EcoRI? Why not?
QIAprep Spin