pEZ – T vector Kit - RNA Inc.-Future Biotechnology … · Web viewpEZ – T vector kit provides a simple, efficient cloning strategy for the direct insertion of a PCR product into

pEZ – T vector Kit

1. Description ----------------------------------------------- 2

2. Components ---------------------------------------------- 2

3. Brief Protocol -------------------------------------------- 3

4. Ordering Information ------------------------------------- 3

5. pEZ – T vector map, MCS sequence ------------------------ 4

6. pEZ – T vector full sequence -------------------------------

5

7. Restriction site ------------------------------------------- 7

8. Troubleshooting ------------------------------------------ 10

Future Biotechnology System – RNA inc. 1

www. RNA. com

Description

pEZ – T vector kit provides a simple, efficient cloning strategy for the direct insertion of a PCR product into a plasmid vector.

- Convenient EcoR I, Nco I restriction sites flanking the PCR product insertion site for easy excision of inserts

- T7, T3 promoter / primer site for in vitro RNA transcription and sequencing

- M13 forward and reverse primer sites for sequencing or PCR screening

- Multiple cloning site in the forward (+) and reverse (-) orientations for easier cloning

pEZ – T vector 5’-overhang (Xho I, Sal I, Cla I, Mlu I, EcoR I, Nco I, BamH I, Spe I, Xba I, Not I)

3’-overhang (Kpn I, Apa I, Hinc II, Pst I, Sac II, Sac I, BstX I)

Blunt-end (Sma I)

Components

- 1.2 ug pEZ – T vector (50 ng/ul)

- 10 ul Ligase (3 u/ul)

- 40 ul Ligase Buffer

- 10 ul Control DNA (5 ng/ul)


www. RNA. com

Brief Protocol

A. Use the formular below to estimate the amount of PCR product needed to l igate with pEZ – T vector

X ng Vector × X bp Insert X Kb vector

× insert : vector molar ratio = X ng insert

ex)50 ng vector × 1 kb insert

3 Kb vector×

31

= 50 ng insert

B. Set up the 10 ul ligation reaction as follows ;Fresh PCR product X ul10 X ligation buffer 1 ulpEZ-T vector 1 ulSterile H2O X ulT4 DNA ligase 0.5 ul

Final volume 10 ul

C. Incubate the ligation reaction at 16℃ for 2 hour or at 4℃ for 12 hour.D. Transformation

Ordering Information

Product Quantity Cat No. Storage condition

pEZ – T vector Kit 20 reaction 101010 6 months after purchase

pEZ – T vector Kit 40 reaction 101011 6 months after purchase

pEZ – T vector 20 reaction 101012 6 months after purchase

pEZ – T vector 40 reaction 101013 6 months after purchase


www. RNA. com

pEZ – T vector

MCS sequence


www. RNA. com

pEZ – T vector full sequence

5' to 3'

ＣＴＡＡＡＴＴＧＴＡ　　ＡＧＣＧＴＴＡＡＴＡ　　ＴＴＴＴＧＴＴＡＡＡ　　ＡＴＴＣＧＣＧＴＴＡ　　ＡＡＴＴＴＴＴＧＴＴ　 50ＡＡＡＴＣＡＧＣＴＣ　　ＡＴＴＴＴＴＴＡＡＣ　　ＣＡＡＴＡＧＧＣＣＧ　　ＡＡＡＴＣＧＧＣＡＡ　　ＡＡＴＣＣＣＴＴＡＴ　 100ＡＡＡＴＣＡＡＡＡＧ　　ＡＡＴＡＧＡＣＣＧＡ　　ＧＡＴＡＧＧＧＴＴＧ　　ＡＧＴＧＴＴＧＴＴＣ　　ＣＡＧＴＴＴＧＧＡＡ　 150ＣＡＡＧＡＧＴＣＣＡ　　ＣＴＡＴＴＡＡＡＧＡ　　ＡＣＧＴＧＧＡＣＴＣ　　ＣＡＡＣＧＴＣＡＡＡ　　ＧＧＧＣＧＡＡＡＡＡ　 200ＣＣＧＴＣＴＡＴＣＡ　　ＧＧＧＣＧＡＴＧＧＣ　　ＣＣＡＣＴＡＣＧＴＧ　　ＡＡＣＣＡＴＣＡＣＣ　　ＣＴＡＡＴＣＡＡＧＴ　 250ＴＴＴＴＴＧＧＧＧＴ　　ＣＧＡＧＧＴＧＣＣＧ　　ＴＡＡＡＧＣＡＣＴＡ　　ＡＡＴＣＧＧＡＡＣＣ　　ＣＴＡＡＡＧＧＧＡＧ 300ＣＣＣＣＣＧＡＴＴＴ　　ＡＧＡＧＣＴＴＧＡＣ　　ＧＧＧＧＡＡＡＧＣＣ　　ＧＧＣＧＡＡＣＧＴＧ　　ＧＣＧＡＧＡＡＡＧＧ　 350ＡＡＧＧＧＡＡＧＡＡ　　ＡＧＣＧＡＡＡＧＧＡ　　ＧＣＧＧＧＣＧＣＴＡ　　ＧＧＧＣＧＣＴＧＧＣ　　ＡＡＧＴＧＴＡＧＣＧ　 400ＧＴＣＡＣＧＣＴＧＣ　　ＧＣＧＴＡＡＣＣＡＣ　　ＣＡＣＡＣＣＣＧＣＣ　　ＧＣＧＣＴＴＡＡＴＧ　　ＣＧＣＣＧＣＴＡＣＡ　 450ＧＧＧＣＧＣＧＴＣＣ　　ＣＡＴＴＣＧＣＣＡＴ　　ＴＣＡＧＧＣＴＧＣＧ　　ＣＡＡＣＴＧＴＴＧＧ　　ＧＡＡＧＧＧＣＧＡＴ　　 500ＣＧＧＴＧＣＧＧＧＣ　　ＣＴＣＴＴＣＧＣＴＡ　　ＴＴＡＣＧＣＣＡＧＣ　　ＴＧＧＣＧＡＡＡＧＧ　　ＧＧＧＡＴＧＴＧＣＴ　　 550ＧＣＡＡＧＧＣＧＡＴ　　ＴＡＡＧＴＴＧＧＧＴ　　ＡＡＣＧＣＣＡＧＧＧ　　ＴＴＴＴＣＣＣＡＧＴ　　ＣＡＣＧＡＣＧＴＴＧ 600ＴＡＡＡＡＣＧＡＣＧ　　ＧＣＣＡＧＴＧＡＧＣ　　ＧＣＧＣＧＴＡＡＴＡ　　ＣＧＡＣＴＣＡＣＴＡ　　ＴＡＧＧＧＣＧＡＡＴ　 650ＴＧＧＡＧＣＴＣＣＡ　　ＣＣＧＣＧＧＴＧＧＣ　　ＧＧＣＣＧＣＴＣＴＡ　　ＧＡＡＣＴＡＧＴＧＧ　　ＡＴＣＣＣＣＣＧＧＧ　 700ＣＴＧＣＡＧＧＡＡＴ　　ＴＣＣＡＴＧＧＡＴＡ　　ＣＣＡＴＧＧＴＧＡＡ　　ＴＴＣＴＡＣＧＣＧＴ　　ＡＧＣＴＴＡＴＣＧＡ 750

ＴＡＣＣＧＴＣＧＡＣＣＴＣＧＡＧＧＧＧＧＧＧＣＣＣＧＧＴＡＣＣＣＡＧＣＴＴＴＴＧＴＴＣＣＣＴＴＴＡＧ 800ＴＧＡＧＧＧＴＴＡＡＴＴＧＣＧＣＧＣＴＴＧＧＣＧＴＡＡＴＣＡＴＧＧＴＣＡＴＡＧＣＴＧＴＴＴＣＣＴＧＴ 850ＧＴＧＡＡＡＴＴＧＴＴＡＴＣＣＧＣＴＣＡＣＡＡＴＴＣＣＡＣＡＣＡＡＣＡＴＡＣＧＡＧＣＣＧＧＡＡＧＣＡ 900ＴＡＡＡＧＴＧＴＡＡＡＧＣＣＴＧＧＧＧＴＧＣＣＴＡＡＴＧＡＧＴＧＡＧＣＴＡＡＣＴＣＡＣＡＴＴＡＡＴＴ 950ＧＣＧＴＴＧＣＧＣＴＣＡＣＴＧＣＣＣＧＣＴＴＴＣＣＡＧＴＣＧＧＧＡＡＡＣＣＴＧＴＣＧＴＧＣＣＡＧＣＴ 1000ＧＣＡＴＴＡＡＴＧＡＡＴＣＧＧＣＣＡＡＣＧＣＧＣＧＧＧＧＡＧＡＧＧＣＧＧＴＴＴＧＣＧＴＡＴＴＧＧＧＣ 1050ＧＣＴＣＴＴＣＣＧＣＴＴＣＣＴＣＧＣＴＣＡＣＴＧＡＣＴＣＧＣＴＧＣＧＣＴＣＧＧＴＣＧＴＴＣＧＧＣＴＧ 1100ＣＧＧＣＧＡＧＣＧＧＴＡＴＣＡＧＣＴＣＡＣＴＣＡＡＡＧＧＣＧＧＴＡＡＴＡＣＧＧＴＴＡＴＣＣＡＣＡＧＡ 1150ＡＴＣＡＧＧＧＧＡＴＡＡＣＧＣＡＧＧＡＡＡＧＡＡＣＡＴＧＴＧＡＧＣＡＡＡＡＧＧＣＣＡＧＣＡＡＡＡＧＧ 1200ＣＣＡＧＧＡＡＣＣＧＴＡＡＡＡＡＧＧＣＣＧＣＧＴＴＧＣＴＧＧＣＧＴＴＴＴＴＣＣＡＴＡＧＧＣＴＣＣＧＣ 1250ＣＣＣＣＣＴＧＡＣＧＡＧＣＡＴＣＡＣＡＡＡＡＡＴＣＧＡＣＧＣＴＣＡＡＧＴＣＡＧＡＧＧＴＧＧＣＧＡＡＡ 1300ＣＣＣＧＡＣＡＧＧＡＣＴＡＴＡＡＡＧＡＴＡＣＣＡＧＧＣＧＴＴＴＣＣＣＣＣＴＧＧＡＡＧＣＴＣＣＣＴＣＧ 1350ＴＧＣＧＣＴＣＴＣＣＴＧＴＴＣＣＧＡＣＣＣＴＧＣＣＧＣＴＴＡＣＣＧＧＡＴＡＣＣＴＧＴＣＣＧＣＣＴＴＴ 1400ＣＴＣＣＣＴＴＣＧＧＧＡＡＧＣＧＴＧＧＣＧＣＴＴＴＣＴＣＡＴＡＧＣＴＣＡＣＧＣＴＧＴＡＧＧＴＡＴＣＴ 1450ＣＡＧＴＴＣＧＧＴＧＴＡＧＧＴＣＧＴＴＣＧＣＴＣＣＡＡＧＣＴＧＧＧＣＴＧＴＧＴＧＣＡＣＧＡＡＣＣＣＣ 1500ＣＣＧＴＴＣＡＧＣＣＣＧＡＣＣＧＣＴＧＣＧＣＣＴＴＡＴＣＣＧＧＴＡＡＣＴＡＴＣＧＴＣＴＴＧＡＧＴＣＣ 1550ＡＡＣＣＣＧＧＴＡＡＧＡＣＡＣＧＡＣＴＴＡＴＣＧＣＣＡＣＴＧＧＣＡＧＣＡＧＣＣＡＣＴＧＧＴＡＡＣＡＧ 1600ＧＡＴＴＡＧＣＡＧＡＧＣＧＡＧＧＴＡＴＧＴＡＧＧＣＧＧＴＧＣＴＡＣＡＧＡＧＴＴＣＴＴＧＡＡＧＴＧＧＴ 1650


www. RNA. com

ＧＧＣＣＴＡＡＣＴＡＣＧＧＣＴＡＣＡＣＴＡＧＡＡＧＧＡＣＡＧＴＡＴＴＴＧＧＴＡＴＣＴＧＣＧＣＴＣＴＧ 1700ＣＴＧＡＡＧＣＣＡＧＴＴＡＣＣＴＴＣＧＧＡＡＡＡＡＧＡＧＴＴＧＧＴＡＧＣＴＣＴＴＧＡＴＣＣＧＧＣＡＡ 1750ＡＣＡＡＡＣＣＡＣＣＧＣＴＧＧＴＡＧＣＧＧＴＧＧＴＴＴＴＴＴＴＧＴＴＴＧＣＡＡＧＣＡＧＣＡＧＡＴＴＡ 1800ＣＧＣＧＣＡＧＡＡＡＡＡＡＡＧＧＡＴＣＴＣＡＡＧＡＡＧＡＴＣＣＴＴＴＧＡＴＣＴＴＴＴＣＴＡＣＧＧＧＧ 1850ＴＣＴＧＡＣＧＣＴＣＡＧＴＧＧＡＡＣＧＡＡＡＡＣＴＣＡＣＧＴＴＡＡＧＧＧＡＴＴＴＴＧＧＴＣＡＴＧＡＧ 1900ＡＴＴＡＴＣＡＡＡＡＡＧＧＡＴＣＴＴＣＡＣＣＴＡＧＡＴＣＣＴＴＴＴＡＡＡＴＴＡＡＡＡＡＴＧＡＡＧＴＴ 1950ＴＴＡＡＡＴＣＡＡＴＣＴＡＡＡＧＴＡＴＡＴＡＴＧＡＧＴＡＡＡＣＴＴＧＧＴＣＴＧＡＣＡＧＴＴＡＣＣＡＡ 2000ＴＧＣＴＴＡＡＴＣＡＧＴＧＡＧＧＣＡＣＣＴＡＴＣＴＣＡＧＣＧＡＴＣＴＧＴＣＴＡＴＴＴＣＧＴＴＣＡＴＣ 2050ＣＡＴＡＧＴＴＧＣＣＴＧＡＣＴＣＣＣＣＧＴＣＧＴＧＴＡＧＡＴＡＡＣＴＡＣＧＡＴＡＣＧＧＧＡＧＧＧＣＴ 2100ＴＡＣＣＡＴＣＴＧＧＣＣＣＣＡＧＴＧＣＴＧＣＡＡＴＧＡＴＡＣＣＧＣＧＡＧＡＣＣＣＡＣＧＣＴＣＡＣＣＧ 2150ＧＣＴＣＣＡＧＡＴＴＴＡＴＣＡＧＣＡＡＴＡＡＡＣＣＡＧＣＣＡＧＣＣＧＧＡＡＧＧＧＣＣＧＡＧＣＧＣＡＧ 2200ＡＡＧＴＧＧＴＣＣＴＧＣＡＡＣＴＴＴＡＴＣＣＧＣＣＴＣＣＡＴＣＣＡＧＴＣＴＡＴＴＡＡＴＴＧＴＴＧＣＣ 2250ＧＧＧＡＡＧＣＴＡＧＡＧＴＡＡＧＴＡＧＴＴＣＧＣＣＡＧＴＴＡＡＴＡＧＴＴＴＧＣＧＣＡＡＣＧＴＴＧＴＴ 2300ＧＣＣＡＴＴＧＣＴＡＣＡＧＧＣＡＴＣＧＴＧＧＴＧＴＣＡＣＧＣＴＣＧＴＣＧＴＴＴＧＧＴＡＴＧＧＣＴＴＣ 2350ＡＴＴＣＡＧＣＴＣＣＧＧＴＴＣＣＣＡＡＣＧＡＴＣＡＡＧＧＣＧＡＧＴＴＡＣＡＴＧＡＴＣＣＣＣＣＡＴＧＴ 2400ＴＧＴＧＣＡＡＡＡＡＡＧＣＧＧＴＴＡＧＣＴＣＣＴＴＣＧＧＴＣＣＴＣＣＧＡＴＣＧＴＴＧＴＣＡＧＡＡＧＴ 2450ＡＡＧＴＴＧＧＣＣＧＣＡＧＴＧＴＴＡＴＣＡＣＴＣＡＴＧＧＴＴＡＴＧＧＣＡＧＣＡＣＴＧＣＡＴＡＡＴＴＣ 2500ＴＣＴＴＡＣＴＧＴＣＡＴＧＣＣＡＴＣＣＧＴＡＡＧＡＴＧＣＴＴＴＴＣＴＧＴＧＡＣＴＧＧＴＧＡＧＴＡＣＴ 2550ＣＡＡＣＣＡＡＧＴＣＡＴＴＣＴＧＡＧＡＡＴＡＧＴＧＴＡＴＧＣＧＧＣＧＡＣＣＧＡＧＴＴＧＣＴＣＴＴＧＣ 2600ＣＣＧＧＣＧＴＣＡＡＴＡＣＧＧＧＡＴＡＡＴＡＣＣＧＣＧＣＣＡＣＡＴＡＧＣＡＧＡＡＣＴＴＴＡＡＡＡＧＴ 2650ＧＣＴＣＡＴＣＡＴＴＧＧＡＡＡＡＣＧＴＴＣＴＴＣＧＧＧＧＣＧＡＡＡＡＣＴＣＴＣＡＡＧＧＡＴＣＴＴＡＣ 2700ＣＧＣＴＧＴＴＧＡＧＡＴＣＣＡＧＴＴＣＧＡＴＧＴＡＡＣＣＣＡＣＴＣＧＴＧＣＡＣＣＣＡＡＣＴＧＡＴＣＴ 2750ＴＣＡＧＣＡＴＣＴＴＴＴＡＣＴＴＴＣＡＣＣＡＧＣＧＴＴＴＣＴＧＧＧＴＧＡＧＣＡＡＡＡＡＣＡＧＧＡＡＧ 2800ＧＣＡＡＡＡＴＧＣＣＧＣＡＡＡＡＡＡＧＧＧＡＡＴＡＡＧＧＧＣＧＡＣＡＣＧＧＡＡＡＴＧＴＴＧＡＡＴＡＣ 2850ＴＣＡＴＡＣＴＣＴＴＣＣＴＴＴＴＴＣＡＡＴＡＴＴＡＴＴＧＡＡＧＣＡＴＴＴＡＴＣＡＧＧＧＴＴＡＴＴＧＴ 2900ＣＴＣＡＴＧＡＧＣＧＧＡＴＡＣＡＴＡＴＴＴＧＡＡＴＧＴＡＴＴＴＡＧＡＡＡＡＡＴＡＡＡＣＡＡＡＴＡＧＧ 2950

ＧＧＴＴＣＣＧＣＧＣＡＣＡＴＴＴＣＣＣＣＧＡＡＡＡＧＴＧＣＣＡＣ 2982


www. RNA. com

Restriction enzymes that cut the pEZ- T vector

Enzyme No.

Cuts of sites Enzyme No.

Cuts of sites

Acc113 IAcc16 IAcc65 IAccB1 IAccBS IAcc IAclN IAcs IAcy IAfl IIIAhd IAlw21 IAlw44 IAlwN IAma87 IApa IApaL IApo IAse IAsn IAsp700 IAsp718 IAspE IAspH IAva IAviI IBamH IBan IBan IIBan IIIBbi IIBbv12 I

12145114121421212433111422143114

2547 479 2289 776 264 776 918 2015 374 678 869 1110 2911 756 683 29 40 707 728 2604 735 1174 2067 657 1492 2653 2738 1488 2734 1590 695 761 774 1488 2734 29 40 707 728 945 1004 2239 945 1004 2239 2666 7762067 657 1492 2653 2738695 761479 2289 689 264 776 918 2015 302 657 774 747 2604 657 1492 2653 27382594

BsaO I BsaW IBsc IBse118 I BseC IBseP IBsh1285 IBshN IBsiE IBsiHKA IBsi IBsoB IBsp106 IBsp120 IBsp143 IIBsp19 IBspC IBspD IBspH IBspLU11 IBspX IBsrB IBsrD IBsrF IBssA IBssH IIBssS IBssT1 IBstD102 IBstDS IBstH2 IBst I

63121264642211422121152222225341

500 673 1090 1514 2437 2586 1380 1527 2358 747 328 2147747 619 813 500 673 1090 1514 2437 2586 264 776 918 2015 500 673 1090 1514 2437 2586 657 1492 2653 2738 1352 2736 695 761 747 770 378 386 1052 1422 712 721 500 2437 747 1894 2902 1174 747 374 678 869 1110 2911 2126 2308 328 2147 328 2147 619 813 1352 2736 712 721 374 678 869 1110 2911 661 712 721 378 386 1052 1422 689


www. RNA. com

Bcg IBco IBgl IBpm IBsa29 IBsaA IBsaH IBsa I

12211111

695 761472 2187 2157 747 227 2604 2139

BstMC IBstSF IBstX2 I

BstX I BstY I

BstZ IBsu15 I

667

17

11

500 673 1090 1514 2437 2586 446 638 701 1439 1630 2308 689 1815 1826 1912 1924 2692 2709665 689 1815 1826 1912 1924 2692 2709670 747

Enzyme No.

Cuts of sites Enzyme No.

Cuts of sites

CciN ICfr10 ICfr42 ICfr9 ICfr ICla IDra IDra IIDra IIIDrd IDsa IEae IEag IEam1104 IEam1105 IEar IEcl136 IIEclHK IEclX IEco130 IEco24 IEco255 IEco31 IEco52 I

121141311234131311123111

670 328 2147 664695609 670 1013 2455 7471933 1952 2644 770 227 182 1282 661 712 721 609 670 1013 2455 670 516 1057 2861 2067 516 1057 2861 655 2067 670 712 721 302 657 774 2547 2139 670 1706 2754

MroN IMsl IMsp17 IMspA1 INae INco INgoA IVNgoM INot INspB IINsp IPaeR7 IPle19 IPshB IPsp124B IPsp1406 IPspA IPspAL IPspOM IPst IPvu IPvu IIRca ISac I

141612111611231211112221

328 663 2319 2478 28372604529 663 998 1516 1761 2702 330 712 721 328 328 670 529 663 998 1516 1761 2702 1178 761 500 2437 945 1004 2239 657 2293 2666 695 697 770 705 500 2437 529 998 1894 2902 657664


Eco57 IEco64 IEco88 IEcoICR IEcoO109 IEcoR IEcoT14 IErh IFriO IFsp IGsu IHae IIHin1 IHinc IIHind IIHsp92 IKpn IKsp632 IKsp IMfl I

Mlu I

24211222321411111317

1

264 776 918 2015 695 761 655 770 707 728 712 721 712 721 302 657 774 479 2289 2157 378 386 1052 1422 2604 757 757 2604 780 516 1057 2861 664 689 1815 1826 1912 1924 2692 2709735

Sac IISal ISap ISca ISfc ISfr274 ISfr303 ISma ISpe ISsp ISst ISst IISty IVne IVsp IXba IXho IXho II

Xma IXma IIIXmn I

111161111211223117

111

755 1057 2547 446 638 701 1439 1630 2308 761664697 68319 2871 657 664 712 721 1488 2734 945 1004 2239 677 761 689 1815 1826 1912 1924 2692 2709695 670 2666

Restriction enzymes that do not cut the pEZ- T vector

Aat IAat IIAccB7 IAcc IIIAfe IAfI IIAge IAoc IAor51H IAsc I

Asp IAts IAvr IIBal IBbe IBbrP IBbs IBbu IBbv16 IIBcI I

Bfr IBgl IIBln IBlp IBpi lBpu1102 IBpu14 IBpuA IBsaB IBsaM I

Bse21 IBse8 IBseA IBseR IBsg IBsh1365 IBsiM IBsiW IBsmB IBsm I

Bsp119 IBsp13 IBsp1407 IBsp1720 IBsp68 IBspE IBspM IBspT IBsrBR IBsrG I

Bst1107 IBst98 IBstB IBstE IIBstP IBstSN IBsu36 ICel IICpo ICsp45 I

Csp ICvn IEco105 IEco147 IEco32 IEco47 IIIEco72 IEco81 IEco1 IEcoN I


www. RNA. com

EcoO65 IEcoR V EcoT22 I Ehe I Esp1396 I Esp3 I FauND I Fba I Fse I Hind III

Hpa I Kas I Kpn2 I Ksp22 I Lsp I Mam I Mfe I MluN I Mph1103 I Mro I

Msc I MspC IMun I Mva1269 I Nar I Nde I Nhe I Nru I Nsi I Nsp V

Pac I Pae I Pfl23 II PflM I PinA I PmaC I Pme55 I Pme I Pml I Ppu10 I

PpuM IPshA I Psp5 II PspE I PspL I PstNH I Rsr II Sbf I SexA I Sfi I

Sfu I Sgf I SgrA ISmi I SnaB I Sph I Spl I Srf I Sse8387 I SseB I

SspB IStu I Sun I Swa ITth111 I Van91 I Vha464 I Zsp2 I


Troubleshooting

Sympton Reason Overcome

No colonies

Competent cell have lost competence.

Check the transformation efficiency of the cells.

The plates are too old.Be sure ampicillin plates are fresh.( <1 month old )

Majority of colonies are blue or light blue with

very few white colonies.

The PCR fragment is inserted, but it is not disrupting the lacZ gene

If you have a small insert (<500bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain inserts.

PCR product is not ligating because there are no 3’-A overhangs

Do not use Vent or Pfu

polymerase as they do not add 3’-A overhangs. Use Taq polymerase.

PCR products were gel-purified before ligation. Gel purification can remove the single 3’-A overhangs.

If gel purification is needed, use nuclease-free solutions.

The PCR products were stored for a long period of time before ligation reaction

Use fresh PCR products.Efficiencies are reduced after as little as 1 day of storage.

The high salt content of PCR can inhibit ligation

Use no more than 2-3 ul of the PCR mixture in the ligation reaction.


www. RNA. com

Sympton Reason Overcome

Majority of colonies are blue or light blue with

very few white colonies

Ligation incubation is not long enough

Optimal results are seen with an overnight ligation.

Ligation temperature is too highHigher temperatures (>20 ℃) give rise to increased background and fewer recombinants.

Multiple PCR products are generated

Gel purify the PCR fragment of interest.

The molar ratio of vector : insert in the ligation reaction may be incorrect

Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.

White colonies or blue colonies of normal size are surrounded by smaller, white colonies

The smaller colonies are ampicillin-sensitive satellite colonies. Do not pick the small colonies as they do not contain any plasmid

Be sure the stock solution of ampicillin and your plates are fresh.

White colonies do not grow in liquid culture

These colonies may be ampicillin-sensitive satellite colonies

Be sure to pick large white colonies. Be sure the ampicillin is fresh.


www. RNA. com

Documents

pEZ – T vector Kit - RNA Inc.-Future Biotechnology … · Web viewpEZ – T vector kit provides a simple, efficient cloning strategy for the direct insertion of a PCR product into