User:Lu Wang/Notebook/Team Allergy/2010/07/27

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2010/07/27 Entry for User:Lu_Wang/Notebook/Team_Allergy)
(Entry title)
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-
==Entry title==
+
==Procedures==
-
* Insert content here...
+
 +
Yesterday, we tried to run a diagnostic digest of the Sense + Intron hpRNA. The gel did not work properly, so we are redoing the gel today. We'll also go ahead and ligate antisense parts into all 38 minipreps that we HOPE contains correct Sense + Intron. Once the gel finishes running, we can go back and dispose of the ligations today that do not contain the correct S+I parts.
 +
 +
Today, we are also going to redo the ligation of Sense + Intron for PDK colonies because the gel that we are redoing today suggests that no PDK worked from last week's ligation.
 +
 +
Procedures
 +
#Diagnostic Digest
 +
#Diagnostic Gel
 +
#Ligation of Antisense Part into 38 minipreps
 +
 +
Redoing ligations for Sense + PDK
 +
#Digest of pLTP, pBet v1, pGer with S, P
 +
#PCR purification
 +
#Ligation of Sense+PDK
 +
#Transform and Plate
 +
 +
==Results==
 +
 +
'''Diagnostic Digest'''
 +
 +
38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst
 +
 +
'''Diagnostic Gel'''
 +
 +
From the digest, we can see that two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)
 +
 +
Lanes are:
 +
1-6: LTP + PDK  (3200 & 1200);
 +
 +
7-16: LTP + PAL (3200 & 540);
 +
 +
17,18: Bet1 + PDK (3200 & 1200);
 +
 +
19-22: Bet1 + PAL (3200 & 540);
 +
 +
23,24: Bet2+PDK (3200 & 1200);
 +
 +
25-28: Bet2 + PAL (3200 & 540);
 +
 +
29-32: Ger3 + PDK (3200 & 1200);
 +
 +
33-38: Ger3 + PAL (3200 & 540);
 +
 +
Image: Ladder (every 6)
 +
 +
[[Image: Ddigestrun2.jpg|240px]]
 +
 +
[[Image: Ddigestrun3.jpg|240px]]
 +
 +
Rerun: [[Image:2-assembly-diagnost-L.jpg|50px]] [[Image:2-assembly-diagnost-R.jpg|50px]]
 +
 +
 +
''Antisense-Intron Backbone Digestion''
 +
 +
{| border="1"
 +
!  !! 1-39
 +
|-
 +
! DNA
 +
| align="center" |~1.5 ug
 +
|-
 +
! 10x BSA
 +
| align="center"  | 3
 +
|-
 +
! diH<sub>2</sub>O
 +
| align="center"  |Fill to 30
 +
|-
 +
! Spe
 +
| align="center"  | 1.5
 +
|-
 +
! Pst1
 +
| align="center" | 1.5
 +
|-
 +
!Neb Buffer 2 (10x)
 +
| align="center" | 3
 +
|}
 +
 +
(Let it go for 2 hours and then phosphatized backbone (20 min)
 +
 +
*Ligated with antisense parts and transformed
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 11:28, 30 July 2010

Project name Main project page
Previous entry      Next entry

Procedures

Yesterday, we tried to run a diagnostic digest of the Sense + Intron hpRNA. The gel did not work properly, so we are redoing the gel today. We'll also go ahead and ligate antisense parts into all 38 minipreps that we HOPE contains correct Sense + Intron. Once the gel finishes running, we can go back and dispose of the ligations today that do not contain the correct S+I parts.

Today, we are also going to redo the ligation of Sense + Intron for PDK colonies because the gel that we are redoing today suggests that no PDK worked from last week's ligation.

Procedures

  1. Diagnostic Digest
  2. Diagnostic Gel
  3. Ligation of Antisense Part into 38 minipreps

Redoing ligations for Sense + PDK

  1. Digest of pLTP, pBet v1, pGer with S, P
  2. PCR purification
  3. Ligation of Sense+PDK
  4. Transform and Plate

Results

Diagnostic Digest

38 digests of the sense+intron miniprepped plasmid DNA. The digest was done with Slow Digest Eco and Pst

Diagnostic Gel

From the digest, we can see that two LTPS+Pal worked (9,11), 3 Ger3S+Pal worked (36-38), 4 Bet2S+Pal Worked (25-28)

Lanes are: 1-6: LTP + PDK (3200 & 1200);

7-16: LTP + PAL (3200 & 540);

17,18: Bet1 + PDK (3200 & 1200);

19-22: Bet1 + PAL (3200 & 540);

23,24: Bet2+PDK (3200 & 1200);

25-28: Bet2 + PAL (3200 & 540);

29-32: Ger3 + PDK (3200 & 1200);

33-38: Ger3 + PAL (3200 & 540);

Image: Ladder (every 6)

Rerun:


Antisense-Intron Backbone Digestion

1-39
DNA ~1.5 ug
10x BSA 3
diH2O Fill to 30
Spe 1.5
Pst1 1.5
Neb Buffer 2 (10x) 3

(Let it go for 2 hours and then phosphatized backbone (20 min)

  • Ligated with antisense parts and transformed


Personal tools